GPCR binding proteins and synthesis thereof

ABSTRACT

Provided herein are methods and compositions relating to G protein-coupled receptor (GPCR) libraries having nucleic acids encoding for a scaffold comprising a GPCR binding domain. Libraries described herein include variegated libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries generated when the nucleic acid libraries are translated. Further described herein are cell libraries expressing variegated nucleic acid libraries described herein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Patent Application No. 62/556,863 filed on Sep. 11, 2017 which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 20, 2018, is named 44854-741_201_SL.txt and is 943,473 bytes in size.

BACKGROUND

G protein-coupled receptors (GPCRs) are implicated in a wide variety of diseases. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. Thus, there is a need for improved agents for therapeutic intervention which target GPCRs.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF SUMMARY

Provided herein are antibodies comprising a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2420 to 2436. Provided herein are antibodies comprising a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2420 to 2436; and wherein the antibody is a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv), a single chain antibody, a Fab fragment, a F(ab′)2 fragment, a Fd fragment, a Fv fragment, a single-domain antibody, an isolated complementarity determining region (CDR), a diabody, a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. Provided herein are antibodies wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Provided herein are antibodies, wherein the VL domain is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, or IGLV2-14. Provided herein are methods of inhibiting GLP1R activity, comprising administering the antibodies as described herein. Provided herein are methods for treatment of a metabolic disorder, comprising administering to a subject in need thereof the antibodies as described herein. In some instances, the antibody comprises a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2420 to 2436. Provided herein are methods for treatment of a metabolic disorder, wherein the metabolic disorder is Type II diabetes, or obesity. Provided herein are nucleic acids encoding for a protein comprising a sequence of any one of SEQ ID NOS: 2420 to 2436.

Provided herein are nucleic acid libraries comprising a plurality of nucleic acids, wherein each nucleic acid encodes for a sequence that when translated encodes for an immunoglobulin scaffold, wherein the immunoglobulin scaffold comprises a CDR-H3 loop that comprises a GPCR binding domain, and wherein each nucleic acid comprises a sequence encoding for a sequence variant of the GPCR binding domain. Provided herein are nucleic acid libraries, wherein a length of the CDR-H3 loop is about 20 to about 80 amino acids. Provided herein are nucleic acid libraries, wherein a length of the CDR-H3 loop is about 80 to about 230 base pairs. Provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold further comprises one or more domains selected from variable domain, light chain (VL), variable domain, heavy chain (VH), constant domain, light chain (CL), and constant domain, heavy chain (CH). Provided herein are nucleic acid libraries, wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Provided herein are nucleic acid libraries, wherein the VL domain is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, or IGLV2-14. Provided herein are nucleic acid libraries, wherein a length of the VH domain is about 90 to about 100 amino acids. Provided herein are nucleic acid libraries, wherein a length of the VL domain is about 90 to about 120 amino acids. Provided herein are nucleic acid libraries, wherein a length of the VH domain is about 280 to about 300 base pairs. Provided herein are nucleic acid libraries, wherein a length of the VL domain is about 300 to about 350 base pairs. Provided herein are nucleic acid libraries, wherein the library comprises at least 105 non-identical nucleic acids. Provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold comprises a single immunoglobulin domain. Provided herein are nucleic acid libraries, wherein the immunoglobulin scaffold comprises a peptide of at most 100 amino acids. Provided herein are vector libraries comprising nucleic acid libraries as described herein. Provided herein are cell libraries comprising nucleic acid libraries as described herein.

Provided herein are nucleic acid libraries comprising a plurality of nucleic acids, wherein each nucleic acid encodes for a sequence that when translated encodes a GPCR binding domain, and wherein each nucleic acid comprises sequence encoding for a different GPCR binding domain about 20 to about 80 amino acids. Provided herein are nucleic acid libraries, wherein a length of the GPCR binding domain is about 80 to about 230 base pairs. Provided herein are nucleic acid libraries, wherein the GPCR binding domain is designed based on conformational ligand interactions, peptide ligand interactions, small molecule ligand interactions, extracellular domains of GPCRs, or antibodies that target GPCRs. Provided herein are vector libraries comprising nucleic acid libraries as described herein. Provided herein are cell libraries comprising nucleic acid libraries as described herein.

Provided herein are protein libraries comprising a plurality of proteins, wherein each of the proteins of the plurality of proteins comprise an immunoglobulin scaffold, wherein the immunoglobulin scaffold comprises a CDR-H3 loop that comprises a sequence variant of a GPCR binding domain. Provided herein are protein libraries, wherein a length of the CDR-H3 loop is about 20 to about 80 amino acids. Provided herein are protein libraries, wherein the immunoglobulin scaffold further comprises one or more domains selected from variable domain, light chain (VL), variable domain, heavy chain (VH), constant domain, light chain (CL), and constant domain, heavy chain (CH). Provided herein are protein libraries, wherein the VH domain is IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV3-74, IGHV4-39, or IGHV4-59/61. Provided herein are protein libraries, wherein the VL domain is IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, or IGLV2-14. Provided herein are protein libraries, wherein a length of the VH domain is about 90 to about 100 amino acids. Provided herein are protein libraries, wherein a length of the VL domain is about 90 to about 120 amino acids. Provided herein are protein libraries, wherein the plurality of proteins is used to generate a peptidomimetic library. Provided herein are protein libraries, wherein the protein library comprises peptides. Provided herein are protein libraries, wherein the protein library comprises immunoglobulins. Provided herein are protein libraries, wherein the protein library comprises antibodies. Provided herein are cell libraries comprising protein libraries as described herein.

Provided herein are protein libraries comprising a plurality of proteins, wherein the plurality of proteins comprises sequence encoding for different GPCR binding domains, and wherein the length of each GPCR binding domain is about 20 to about 80 amino acids. Provided herein are protein libraries, wherein the protein library comprises peptides. Provided herein are protein libraries, wherein the protein library comprises immunoglobulins. Provided herein are protein libraries, wherein the protein library comprises antibodies. Provided herein are protein libraries, wherein the plurality of proteins are used to generate a peptidomimetic library. Provided herein are cell libraries comprising protein libraries as described herein.

Provided herein are vector libraries comprising a nucleic acid library described herein. Provided herein are cell libraries comprising a nucleic acid library described herein. Provided herein are cell libraries comprising a protein library described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic of G protein-coupled receptor (GPCR) ligand interaction surfaces.

FIG. 2A depicts a first schematic of an immunoglobulin scaffold.

FIG. 2B depicts a second schematic of an immunoglobulin scaffold.

FIG. 3 depicts a schematic of a motif for placement in a scaffold.

FIG. 4 depicts a schematic of a GPCR.

FIG. 5 depicts schematics of segments for assembly of clonal fragments and non-clonal fragments.

FIG. 6 depicts schematics of segments for assembly of clonal fragments and non-clonal fragments.

FIG. 7 presents a diagram of steps demonstrating an exemplary process workflow for gene synthesis as disclosed herein.

FIG. 8 illustrates an example of a computer system.

FIG. 9 is a block diagram illustrating an architecture of a computer system.

FIG. 10 is a diagram demonstrating a network configured to incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants, and Network Attached Storage (NAS).

FIG. 11 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIGS. 12A-12C depict sequences of immunoglobulin scaffolds. FIG. 12A discloses SEQ ID NOS 2437-2453, 2448, 2454-2464, 2448, 2465-2471, 2466, 2472-2483, 2462, 2484-2485, 2448, 2486-2496, 2491, 2497-2507, 2491, 2508-2518, 2513, 2519-2520, 2462, 2521-2522, 2491, 2523-2539, 2534, 2540-2544, 2534, 2545-2549, 2534, 2550-2554, 2491, 2555-2559, 2513, 2560, 2556, 2561-2562, 2496, 2563-2564, 2556, 2565-2567, 2491, 2568-2575, 2552, 2576-2577, 2563, 2578-2581, 2522, 2491, 2582-2585, 2567 and 2563, respectively, in order of appearance. FIG. 12B discloses SEQ ID NOS 2586-2620, 2615, 2621-2631, 2459, 2632-2654, 2448, 2655-2658, 2464, 2448, 2659-2661, 2658, 2662-2672, 2658, 2673-2718, 2657, 2719, 2715, 2720-2728, 2720, 2729-2745, 2734, 2746-2753, 2748-2749 and 2754-2755, respectively, in order of appearance. FIG. 12C discloses SEQ ID NOS 2756-2766, 2761, 2767-2785, 2461, 2786-2788, 2448, 2789-2793, 2459, 2794-2798, 2448, 2799-2803, 2448, 2804-2808, 2734, 2809-2831, 2814, 2832-2836, 2820, 2837, 2551, 2838-2843, 2817, 2844, 2522, 2491, 2845-2848, 2522, 2849-2854, 2491, 2855-2859, 2513, 2860-2864, 2448, 2865-2874, 2464, 2448, 2875-2879, 2459, 2880-2881, 2873-2874, 2464, 2448, 2882-2903, 2608 and 2904, respectively, in order of appearance.

FIG. 13 depicts sequences of G protein-coupled receptors scaffolds. FIG. 13 discloses SEQ ID NOS 2905-2911, 2911, 2911-2912, 2912, 2911, 2911, 2911, 2911, 2911-2912, 2912, 2912, 2911, 2911, 2911-2912, 2911-2912, 2912, 2911, 2911, 2913-2915, 2915-2918, 2918, 2918, 2918, 2918, 2918 and 2918, respectively, in order of appearance.

FIG. 14 is a graph of normalized reads for a library for variable domain, heavy chains.

FIG. 15 is a graph of normalized reads for a library for variable domain, light chains.

FIG. 16 is a graph of normalized reads for a library for heavy chain complementarity determining region 3.

FIG. 17A is a plot of light chain frameworks assayed for folding. FIG. 17A discloses SEQ ID NOS 2919-2924, 2712, 2925-2926, 2725, 2927-2932, 2461, 2933, 2455, 2934-2939, 2730, 2742, 2940-2941, 2556, 2942, 2851, 2498, 2943, 2843, 2944-2945, 2757, 2946-2947, 2774 and 2948, respectively, in order of appearance.

FIG. 17B is a plot of light chain frameworks assayed for thermostability. FIG. 17B discloses SEQ ID NOS 2919-2924, 2712, 2925-2926, 2725, 2927-2932, 2461, 2933, 2455, 2934-2939, 2730, 2742, 2940-2941, 2556, 2942, 2851, 2498, 2943, 2843, 2944-2945, 2757, 2946-2947, 2774 and 2948, respectively, in order of appearance.

FIG. 17C is a plot of light chain frameworks assayed for motif display using FLAG tag. FIG. 17C discloses SEQ ID NOS 2919-2924, 2712, 2925-2926, 2725, 2927-2932, 2461, 2933, 2455, 2934-2939, 2730, 2742, 2940-2941, 2556, 2942, 2851, 2498, 2943, 2843, 2944-2945, 2757, 2946-2947, 2774 and 2948, respectively, in order of appearance.

FIG. 17D is a plot of light chain frameworks assayed for motif display using His tag. FIG. 17D discloses SEQ ID NOS 2919-2924, 2712, 2925-2926, 2725, 2927-2932, 2461, 2933, 2455, 2934-2939, 2730, 2742, 2940-2941, 2556, 2942, 2851, 2498, 2943, 2843, 2944-2945, 2757, 2946-2947, 2774 and 2948, respectively, in order of appearance.

FIG. 18A is a plot of heavy chain frameworks assayed for folding.

FIG. 18B is a plot of heavy chain frameworks assayed for stability.

FIG. 18C is a plot of heavy chain frameworks assayed for motif display using FLAG tag.

FIG. 18D is a plot of heavy chain frameworks assayed for motif display using His tag.

FIG. 18E is a plot of heavy chain frameworks assayed for expression.

FIG. 18F is a plot of heavy chain frameworks assayed for selection specificity.

FIGS. 19A-19C depict images of G protein-coupled receptors visualized by fluorescent antibodies.

FIGS. 20A-20C depict images of G protein-coupled receptors visualized by auto-fluorescent proteins.

FIG. 21A depicts a schematic of an immunoglobulin scaffold comprising a VH domain attached to a VL domain using a linker.

FIG. 21B depicts a schematic of a full-domain architecture of an immunoglobulin scaffold comprising a VH domain attached to a VL domain using a linker, a leader sequence, and pIII sequence.

FIG. 21C depicts a schematic of four framework elements (FW1, FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements for a VL or VH domain.

DETAILED DESCRIPTION

The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques, which are within the skill of the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

Definitions

Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.

GPCR Libraries

Provided herein are methods and compositions relating to G protein-coupled receptor (GPCR) binding libraries comprising nucleic acids encoding for a scaffold comprising a GPCR binding domain. Scaffolds as described herein can stably support a GPCR binding domain. The GPCR binding domain may be designed based on surface interactions of a GPCR ligand and the GPCR. Libraries as described herein may be further variegated to provide for variant libraries comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. Further described herein are protein libraries that may be generated when the nucleic acid libraries are translated. In some instances, nucleic acid libraries as described herein are transferred into cells to generate a cell library. Also provided herein are downstream applications for the libraries synthesized using methods described herein. Downstream applications include identification of variant nucleic acids or protein sequences with enhanced biologically relevant functions, e.g., improved stability, affinity, binding, functional activity, and for the treatment or prevention of a disease state associated with GPCR signaling.

Scaffold Libraries

Provided herein are libraries comprising nucleic acids encoding for a scaffold, wherein sequences for GPCR binding domains are placed in the scaffold. Scaffold described herein allow for improved stability for a range of GPCR binding domain encoding sequences when inserted into the scaffold, as compared to an unmodified scaffold. Exemplary scaffolds include, but are not limited to, a protein, a peptide, an immunoglobulin, derivatives thereof, or combinations thereof. In some instances, the scaffold is an immunoglobulin. Scaffolds as described herein comprise improved functional activity, structural stability, expression, specificity, or a combination thereof. In some instances, scaffolds comprise long regions for supporting a GPCR binding domain.

Provided herein are libraries comprising nucleic acids encoding for a scaffold, wherein the scaffold is an immunoglobulin. In some instances, the immunoglobulin is an antibody. As used herein, the term antibody will be understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CH1 domains), a F(ab′)2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CH1 fragment), a Fv fragment (including fragments comprising the VL and VH domains of a single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti-idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for a scaffold, wherein the scaffold is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for a scaffold, wherein the scaffold is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

Libraries described herein comprising nucleic acids encoding for a scaffold, wherein the scaffold is an immunoglobulin, comprise variations in at least one region of the immunoglobulin. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDR-H1, CDR-H2, and CDR-H3. In some instances, the CDR is a light domain including, but not limited to, CDR-L1, CDR-L2, and CDR-L3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for a scaffold, wherein each nucleic acid encodes for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the scaffold library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons of framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

In some instances, the at least one region of the immunoglobulin for variation is from heavy chain V-gene family, heavy chain D-gene family, heavy chain J-gene family, light chain V-gene family, or light chain J-gene family. In some instances, the light chain V-gene family comprises immunoglobulin kappa (IGK) gene or immunoglobulin lambda (IGL). Exemplary genes include, but are not limited to, IGHV1-18, IGHV1-69, IGHV1-8, IGHV3-21, IGHV3-23, IGHV3-30/33rn, IGHV3-28, IGHV1-69, IGHV3-74, IGHV4-39, IGHV4-59/61, IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, and IGLV2-14.

Provided herein are libraries comprising nucleic acids encoding for immunoglobulin scaffolds, wherein the libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, or VH domain. In some instances, the fragments comprise framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the scaffold libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

Libraries comprising nucleic acids encoding for immunoglobulin scaffolds as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 amino acids to about 75 amino acids. In some instances, the immunoglobulin scaffolds comprise at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than 5000 amino acids.

A number of variant sequences for the at least one region of the immunoglobulin for variation are de novo synthesized using methods as described herein. In some instances, a number of variant sequences is de novo synthesized for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or combinations thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is at least or about 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or more than 8000 sequences. In some instances, the number of variant sequences is about 10 to 500, 25 to 475, 50 to 450, 75 to 425, 100 to 400, 125 to 375, 150 to 350, 175 to 325, 200 to 300, 225 to 375, 250 to 350, or 275 to 325 sequences.

Variant sequences for the at least one region of the immunoglobulin, in some instances, vary in length or sequence. In some instances, the at least one region that is de novo synthesized is for CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or combinations thereof. In some instances, the at least one region that is de novo synthesized is for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 variant nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 additional nucleotides or amino acids as compared to wild-type. In some instances, the variant sequence comprises at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 less nucleotides or amino acids as compared to wild-type. In some instances, the libraries comprise at least or about 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ variants.

Following synthesis of scaffold libraries, scaffold libraries may be used for screening and analysis. For example, scaffold libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. In some instances, scaffold libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

In some instances, the scaffold libraries are assayed for functional activity, structural stability (e.g., thermal stable or pH stable), expression, specificity, or a combination thereof. In some instances, the scaffold libraries are assayed for scaffolds capable of folding. In some instances, a region of the antibody is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof. For example, a VH region or VL region is assayed for functional activity, structural stability, expression, specificity, folding, or a combination thereof.

GPCR Libraries

Provided herein are G protein-coupled receptor (GPCR) binding libraries comprising nucleic acids encoding for scaffolds comprising sequences for GPCR binding domains. In some instances, the scaffolds are immunoglobulins. In some instances, the scaffolds comprising sequences for GPCR binding domains are determined by interactions between the GPCR binding domains and the GPCRs.

Provided herein are libraries comprising nucleic acids encoding scaffolds comprising GPCR binding domains, wherein the GPCR binding domains are designed based on surface interactions on the GPCRs. Exemplary GPCRs are seen in Table 1. In some instances, the GPCR binding domains interact with the amino- or carboxy-terminus of the GPCR. In some instances, the GPCR binding domains interact with at least one transmembrane domain including, but not limited to, transmembrane domain 1 (TM1), transmembrane domain 2 (TM2), transmembrane domain 3 (TM3), transmembrane domain 4 (TM4), transmembrane domain 5 (TM5), transmembrane domain 6 (TM6), and transmembrane domain 7 (TM7). In some instances, the GPCR binding domains interact with an intracellular surface of the GPCR. For example, the GPCR binding domains interact with at least one intracellular loop including, but not limited to, intracellular loop 1 (ICL1), intracellular loop 2 (ICL2), and intracellular loop 3 (ICL3). In some instances, the GPCR binding domains interact with an extracellular surface of the GPCR. See FIG. 1. For example, the GPCR binding domains interact with at least one extracellular domain (ECD) or extracellular loop (ECL) of the GPCR. The extracellular loops include, but are not limited to, extracellular loop 1 (ECL1), extracellular loop 2 (ECL2), and extracellular loop 3 (ECL3).

TABLE 1 List of GPCRs Accession GPCR Gene Name Number 5-hydroxytryptamine receptor 1A HTR1A P08908 5-hydroxytryptamine receptor 1B HTR1B P28222 5-hydroxytryptamine receptor 1D HTR1D P28221 5-hydroxytryptamine receptor 1E HTR1E P28566 5-hydroxytryptamine receptor 1F HTR1F P30939 5-hydroxytryptamine receptor 2A HTR2A P28223 5-hydroxytryptamine receptor 2B HTR2B P41595 5-hydroxytryptamine receptor 2C HTR2C P28335 5-hydroxytryptamine receptor 4 HTR4 Q13639 5-hydroxytryptamine receptor 5A HTR5A P47898 5-hydroxytryptamine receptor 6 HTR6 P50406 5-hydroxytryptamine receptor HTR7 P34969 Adenosine receptor A1 ADORA1 P30542 Adenosine receptor A2a ADORA2A P29274 Adenosine receptor A2b ADORA2B P29275 Adenosine A3 receptor ADORA3 P33765 Muscarinic acetylcholine receptor M1 CHRM1 P11229 Muscarinic acetylcholine receptor M2 CHRM2 P08172 Muscarinic acetylcholine receptor M3 CHRM3 P20309 Muscarinic acetylcholine receptor M4 CHRM4 P08173 Muscarinic acetylcholine receptor M5 CHRM5 P08912 Adrenocorticotropic hormone receptor MC2R Q01718 α-1A adrenergic receptor ADRA1A P35348 α-1B adrenergic receptor ADRA1B P35368 α-1D adrenergic receptor ADRA1D P25100 α-2A adrenergic receptor ADRA2A P08913 α-2B adrenergic receptor ADRA2B P18089 α-2C adrenergic receptor ADRA2C P18825 β-1 adrenergic receptor ADRB1 P08588 β-2 adrenergic receptor ADRB2 P07550 β-3 adrenergic receptor ADRB3 P13945 Type-1 angiotensin II receptor AGTR1 P30556 Duffy antigen/chemokine receptor DARC Q16570 Endothelin-1 receptor EDNRA P25101 Endothelin B receptor EDNRB P24530 N-formyl peptide receptor 2 FPR2 P25090 Follicle-stimulating hormone receptor FSHR P23945 Galanin receptor type 1 GALR1 P47211 Galanin receptor type 2 GALR2 O43603 Galanin receptor type 3 GALR3 O60755 Gastrin/cholecystokinin type B receptor CCKBR P32239 Gonadotropin-releasing hormone receptor GNRHR P30968 Putative gonadotropin-releasing hormone II GNRHR2 Q96P88 receptor G-protein coupled oestrogen receptor 1 GPER Q99527 Uracil nucleotide/cysteinyl leukotriene GPR17 Q13304 receptor Putative G-protein coupled receptor 44 GPR44 Q9Y5Y4 G-protein coupled receptor 55 GPR55 Q9Y2T6 Gastrin-releasing peptide receptor GRPR P30550 Histamine H1 receptor HRH1 P35367 Histamine H2 receptor HRH2 P25021 Histamine H3 receptor HRH3 Q9Y5N1 Histamine H4 receptor HRH4 Q9H3N8 KiSS-1 receptor KISS1R Q969F8 Lysophosphatidic acid receptor 1 LPAR1 Q92633 Lysophosphatidic acid receptor 2 LPAR2 Q9HBW0 Lysophosphatidic acid receptor 3 LPAR3 Q9UBY5 Lysophosphatidic acid receptor 4 LPAR4 Q99677 Lysophosphatidic acid receptor 6 LPAR6 P43657 Lutropin-choriogonadotropic hormone LHCGR P22888 receptor Leukotriene B4 receptor 1 LTB4R Q15722 Leukotriene B4 receptor 2 LTB4R2 Q9NPC1 Melanocortin receptor 3 MC3R P41968 Melanocortin receptor 4 MC4R P32245 Melanocortin receptor 5 MC5R P33032 Olfactory receptor 10G9 OR10G9 Q8NGN4 Olfactory receptor 10H1 OR10H1 Q9Y4A9 Olfactory receptor 10H2 OR10H2 O60403 Olfactory receptor 10H3 OR10H3 O60404 Olfactory receptor 10H4 OR10H4 Q8NGA5 Olfactory receptor 10H5 OR10H5 Q8NGA6 Olfactory receptor 10J1 OR10J1 P30954 Olfactory receptor 10J3 OR10J3 Q5JRS4 Olfactory receptor 10J5 OR10J5 Q8NHC4 Olfactory receptor 10K1 OR10K1 Q8NGX5 Olfactory receptor 10K2 OR10K2 Q6IF99 Olfactory receptor 10P1 OR10P1 Q8NGE3 Olfactory receptor 10Q1 OR10Q1 Q8NGQ4 Olfactory receptor 10R2 OR10R2 Q8NGX6 Olfactory receptor 10S1 OR10S1 Q8NGN2 Olfactory receptor 10T2 OR10T2 Q8NGX3 Olfactory receptor 10V1 OR10V1 Q8NGI7 Olfactory receptor 10W1 OR10W1 Q8NGF6 Olfactory receptor 14A2 OR14A2 Q96R54 Olfactory receptor 14C36 OR14C36 Q8NHC7 Olfactory receptor 14I1 OR14I1 A6ND48 Olfactory receptor 14J1 OR14J1 Q9UGF5 Olfactory receptor 14K1 OR14K1 Q8NGZ2 Olfactory receptor 2A12 OR2A12 Q8NGT7 Olfactory receptor 2A14 OR2A14 Q96R47 Olfactory receptor 2A25 OR2A25 A4D2G3 Olfactory receptor 2AG1 OR2AG1 Q9H205 Olfactory receptor 2AG2 OR2AG2 A6NM03 Olfactory receptor 2AJ1 OR2AJ1 Q8NGZ0 Olfactory receptor 2AK2 OR2AK2 Q8NG84 Olfactory receptor 2AP1 OR2AP1 Q8NGE2 Olfactory receptor 2AT4 OR2AT4 A6NND4 Olfactory receptor 51I2 OR51I2 Q9H344 Olfactory receptor 51J1 OR51J1 Q9H342 Olfactory receptor 51L1 OR51L1 Q8NGJ5 Olfactory receptor 51M1 OR51M1 Q9H341 Olfactory receptor 51Q1 OR51Q1 Q8NH59 Olfactory receptor 51S1 OR51S1 Q8NGJ8 Olfactory receptor 51T1 OR51T1 Q8NGJ9 Olfactory receptor 51V1 OR51V1 Q9H2C8 Olfactory receptor 52A1 OR52A1 Q9UKL2 Olfactory receptor 52A5 OR52A5 Q9H2C5 Olfactory receptor 52B2 OR52B2 Q96RD2 Olfactory receptor 52B4 OR52B4 Q8NGK2 Olfactory receptor 52B6 OR52B6 Q8NGF0 Olfactory receptor 52D1 OR52D1 Q9H346 Olfactory receptor 52E2 OR52E2 Q8NGJ4 Olfactory receptor 52E4 OR52E4 Q8NGH9 Olfactory receptor 52E5 OR52E5 Q8NH55 Olfactory receptor 52E6 OR52E6 Q96RD3 Olfactory receptor 52E8 OR52E8 Q6IFG1 Olfactory receptor 52H1 OR52H1 Q8NGJ2 Olfactory receptor 52I1 OR52I1 Q8NGK6 Olfactory receptor 52I2 OR52I2 Q8NH67 Olfactory receptor 52K1 OR52K1 Q8NGK4 Olfactory receptor 52K2 OR52K2 Q8NGK3 Olfactory receptor 52L1 OR52L1 Q8NGH7 Olfactory receptor 52M1 OR52M1 Q8NGK5 Olfactory receptor 52N1 OR52N1 Q8NH53 Olfactory receptor 52N2 OR52N2 Q8NGI0 Olfactory receptor 52N4 OR52N4 Q8NGI2 Olfactory receptor 52N5 OR52N5 Q8NH56 Olfactory receptor 52R1 OR52R1 Q8NGF1 Olfactory receptor 52W1 OR52W1 Q6IF63 Red-sensitive opsin OPN1LW P04000 Visual pigment-like receptor peropsin RRH O14718 Olfactory receptor 1A1 OR1A1 Q9P1Q5 Olfactory receptor 1A2 OR1A2 Q9Y585 Olfactory receptor 1B1 OR1B1 Q8NGR6 Olfactory receptor 1C1 OR1C1 Q15619 Olfactory receptor 1D2 OR1D2 P34982 Olfactory receptor 1F1 OR1F1 O43749 Olfactory receptor 1F12 OR1F12 Q8NHA8 Olfactory receptor 1G1 OR1G1 P47890 Olfactory receptor 1I1 OR1I1 O60431 Olfactory receptor 1J1 OR1J1 Q8NGS3 Olfactory receptor 1J2 OR1J2 Q8NGS2 Olfactory receptor 1J4 OR1J4 Q8NGS1 Olfactory receptor 1K1 OR1K1 Q8NGR3 Olfactory receptor 1L1 OR1L1 Q8NH94 Olfactory receptor 1L3 OR1L3 Q8NH93 Olfactory receptor 1L4 OR1L4 Q8NGR5 Olfactory receptor 1L6 OR1L6 Q8NGR2 Olfactory receptor 1L8 OR1L8 Q8NGR8 Olfactory receptor 1M1 OR1M1 Q8NGA1 Olfactory receptor 1N1 OR1N1 Q8NGS0 Olfactory receptor 1N2 OR1N2 Q8NGR9 Olfactory receptor 1Q1 OR1Q1 Q15612 Olfactory receptor 1S1 OR1S1 Q8NH92 Olfactory receptor 1S2 OR1S2 Q8NGQ3 Olfactory receptor 2A2 OR2A2 Q6IF42 Olfactory receptor 2A4 OR2A4 O95047 Olfactory receptor 2B2 OR2B2 Q9GZK3 Putative olfactory receptor 2B3 OR2B3 O76000 Olfactory receptor 2B6 OR2B6 P58173 Putative olfactory receptor 2B8 OR2B8P P59922 Olfactory receptor 2T5 OR2T5 Q6IEZ7 Olfactory receptor 2T6 OR2T6 Q8NHC8 Olfactory receptor 2T8 OR2T8 A6NH00 Olfactory receptor 2V1 OR2V1 Q8NHB1 Olfactory receptor 2V2 OR2V2 Q96R30 Olfactory receptor 2W1 OR2W1 Q9Y3N9 Olfactory receptor 2W3 OR2W3 Q7Z3T1 Olfactory receptor 2Y1 OR2Y1 Q8NGV0 Olfactory receptor 2Z1 OR2Z1 Q8NG97 Olfactory receptor 3A1 OR3A1 P47881 Olfactory receptor 3A2 OR3A2 P47893 Olfactory receptor 3A3 OR3A3 P47888 Olfactory receptor 3A4 OR3A4 P47883 Putative olfactory receptor 4A4 OR4A4P Q8NGN8 Olfactory receptor 4A5 OR4A5 Q8NH83 Olfactory receptor 4A8 OR4A8P P0C604 Olfactory receptor 4B1 OR4B1 Q8NGF8 Olfactory receptor 4C3 OR4C3 Q8NH37 Olfactory receptor 4C5 OR4C5 Q8NGB2 Olfactory receptor 4C6 OR4C6 Q8NH72 Olfactory receptor 4C11 OR4C11 Q6IEV9 Olfactory receptor 4C12 OR4C12 Q96R67 Olfactory receptor 4C13 OR4C13 Q8NGP0 Olfactory receptor 4C15 OR4C15 Q8NGM1 Olfactory receptor 4C16 OR4C16 Q8NGL9 Olfactory receptor 4D1 OR4D1 Q15615 Olfactory receptor 4D2 OR4D2 P58180 Olfactory receptor 4D5 OR4D5 Q8NGN0 Olfactory receptor 4D6 OR4D6 Q8NGJ1 Olfactory receptor 4D9 OR4D9 Q8NGE8 Olfactory receptor 4D10 OR4D10 Q8NGI6 Olfactory receptor 4D11 OR4D11 Q8NGI4 Olfactory receptor 5B17 OR5B17 Q8NGF7 Olfactory receptor 5B21 OR5B21 A6NL26 Olfactory receptor 5C1 OR5C1 Q8NGR4 Olfactory receptor 5D13 OR5D13 Q8NGL4 Olfactory receptor 5D14 OR5D14 Q8NGL3 Olfactory receptor 5D16 OR5D16 Q8NGK9 Olfactory receptor 5D18 OR5D18 Q8NGL1 Olfactory receptor 5F1 OR5F1 O95221 Olfactory receptor 5H1 OR5H1 A6NKK0 Olfactory receptor 5H2 OR5H2 Q8NGV7 Olfactory receptor 5H6 OR5H6 Q8NGV6 Olfactory receptor 5I1 OR5I1 Q13606 Olfactory receptor 5J2 OR5J2 Q8NH18 Olfactory receptor 5K1 OR5K1 Q8NHB7 Olfactory receptor 5K2 OR5K2 Q8NHB8 Olfactory receptor 5K3 OR5K3 A6NET4 Olfactory receptor 5K4 OR5K4 A6NMS3 Olfactory receptor 5L1 OR5L1 Q8NGL2 Olfactory receptor 5L2 OR5L2 Q8NGL0 Olfactory receptor 5M1 OR5M1 Q8NGP8 Olfactory receptor 5M3 OR5M3 Q8NGP4 Olfactory receptor 5M8 OR5M8 Q8NGP6 Olfactory receptor 5M9 OR5M9 Q8NGP3 Olfactory receptor 5M10 OR5M10 Q6IEU7 Olfactory receptor 5M11 OR5M11 Q96RB7 Olfactory receptor 5P2 OR5P2 Q8WZ92 Olfactory receptor 5P3 OR5P3 Q8WZ94 Olfactory receptor 5R1 OR5R1 Q8NH85 Olfactory receptor 5T1 OR5T1 Q8NG75 Olfactory receptor 5T2 OR5T2 Q8NGG2 Olfactory receptor 5T3 OR5T3 Q8NGG3 Olfactory receptor 5W2 OR5W2 Q8NH69 Olfactory receptor 7G3 OR7G3 Q8NG95 Olfactory receptor 8A1 OR8A1 Q8NGG7 Olfactory receptor 8B3 OR8B3 Q8NGG8 Olfactory receptor 8B4 OR8B4 Q96RC9 Olfactory receptor 8B8 OR8B8 Q15620 Olfactory receptor 8B12 OR8B12 Q8NGG6 Olfactory receptor 8D1 OR8D1 Q8WZ84 Olfactory receptor 8D2 OR8D2 Q9GZM6 Olfactory receptor 8D4 OR8D4 Q8NGM9 Orexin receptor type 1 HCRTR1 O43613 Orexin receptor type 2 HCRTR2 O43614 Oxoeicosanoid receptor 1 OXER1 Q8TDS5 Oxytocin receptor OXTR P30559 P2Y purinoceptor 1 P2RY1 P47900 P2Y purinoceptor 2 P2RY2 P41231 P2Y purinoceptor 4 P2RY4 P51582 P2Y purinoceptor 6 P2RY6 Q15077 P2Y purinoceptor 8 P2RY8 Q86VZ1 Putative P2Y purinoceptor 10 P2RY10 O00398 P2Y purinoceptor 11 P2RY11 Q96G91 P2Y purinoceptor 12 P2RY12 Q9H244 P2Y purinoceptor 13 P2RY13 Q9BPV8 P2Y purinoceptor 14 P2RY14 Q15391 Proteinase-activated receptor 1 F2R P25116 Proteinase-activated receptor 2 F2RL1 P55085 Proteinase-activated receptor 3 F2RL2 O00254 Proteinase-activated receptor 4 F2RL3 Q96RI0 Prostaglandin D2 receptor PTGDR Q13258 Prostaglandin E2 receptor EP1 subtype PTGER1 P34995 Prostaglandin E2 receptor EP2 subtype PTGER2 P43116 Prostaglandin E2 receptor EP3 subtype PTGER3 P43115 Prostaglandin E2 receptor EP4 subtype PTGER4 P35408 Type-2 angiotensin II receptor (AT2) AGTR2 P50052 Apelin receptor APLNR P35414 B1 bradykinin receptor BDKRB1 P46663 B2 bradykinin receptor BDKRB2 P30411 C5a anaphylatoxin chemotactic receptor C5AR1 P21730 Cholecystokinin receptor type A CCKAR P32238 C-C chemokine receptor type 10 CCR10 P46092 C-C chemokine receptor type 1 CCR1 P32246 C-C chemokine receptor type 2 CCR2 P41597 C-C chemokine receptor type 3 CCR3 P51677 C-C chemokine receptor type 4 CCR4 P51679 C-C chemokine receptor type 5 CCR5 P51681 C-C chemokine receptor type 6 CCR6 P51684 C-C chemokine receptor type 7 CCR7 P32248 C-C chemokine receptor type 8 CCR8 P51685 C-C chemokine receptor type 9 CCR9 P51686 Cysteinyl leukotriene receptor 1 CYSLTR1 Q9Y271 Cysteinyl leukotriene receptor 2 CYSLTR2 Q9NS75 Cannabinoid receptor 1 CNR1 P21554 Cannabinoid receptor 2 CNR2 P34972 CX3C chemokine receptor 1 CX3CR1 P49238 High affinity interleukin-8 receptor A IL8RA P25024 High affinity interleukin-8 receptor B IL8RB P25025 C-X-C chemokine receptor type 3 CXCR3 P49682 C-X-C chemokine receptor type 4 CXCR4 P61073 C-X-C chemokine receptor type 6 CXCR6 O00574 C-X-C chemokine receptor type 7 CXCR7 P25106 D(1A) dopamine receptor DRD1 P21728 D(2) dopamine receptor DRD2 P14416 D(3) dopamine receptor DRD3 P35462 D(4) dopamine receptor DRD4 P21917 D(1B) dopamine receptor DRD5 P21918 Melanocyte-stimulating hormone receptor MC1R Q01726 Melatonin receptor type 1A MTNR1A P48039 Melatonin receptor type 1B MTNR1B P49286 Substance-P receptor TACR1 P25103 Substance-K receptor TACR2 P21452 Neuromedin-K receptor TACR3 P29371 Neuromedin-B receptor NMBR P28336 Neuropeptides B/W receptor type 1 NPBWR1 P48145 Neuropeptides B/W receptor type 2 NPBWR2 P48146 Neuropeptide FF receptor 1 NPFFR1 Q9GZQ6 Neuropeptide FF receptor 2 NPFFR2 Q9Y5X5 Neuropeptide Y receptor type 1 NPY1R P25929 Neuropeptide Y receptor type 2 NPY2R P49146 Neuropeptide Y receptor type 4 PPYR1 P50391 Neuropeptide Y receptor type 5 NPY5R Q15761 Neurotensin receptor type 1 NTSR1 P30989 Neurotensin receptor type 2 NTSR2 O95665 Olfactory receptor 10A2 OR10A2 Q9H208 Olfactory receptor 10A3 OR10A3 P58181 Olfactory receptor 10A4 OR10A4 Q9H209 Olfactory receptor 10A5 OR10A5 Q9H207 Olfactory receptor 10A6 OR10A6 Q8NH74 Olfactory receptor 10A7 OR10A7 Q8NGE5 Olfactory receptor 10AD1 OR10AD1 Q8NGE0 Olfactory receptor 10AG1 OR10AG1 Q8NH19 Olfactory receptor 10C1 OR10C1 Q96KK4 Olfactory receptor 10G2 OR10G2 Q8NGC3 Olfactory receptor 10G3 OR10G3 Q8NGC4 Olfactory receptor 10G4 OR10G4 Q8NGN3 Olfactory receptor 10G6 OR10G6 Q8NH81 Olfactory receptor 10G7 OR10G7 Q8NGN6 Olfactory receptor 10G8 OR10G8 Q8NGN5 Olfactory receptor 2T10 OR2T10 Q8NGZ9 Olfactory receptor 2T11 OR2T11 Q8NH01 Olfactory receptor 2T12 OR2T12 Q8NG77 Olfactory receptor 2T27 OR2T27 Q8NH04 Olfactory receptor 2T29 OR2T29 Q8NH02 Olfactory receptor 2T33 OR2T33 Q8NG76 Olfactory receptor 2T34 OR2T34 Q8NGX1 Olfactory receptor 2T35 OR2T35 Q8NGX2 Olfactory receptor 4A15 OR4A15 Q8NGL6 Olfactory receptor 4A16 OR4A16 Q8NH70 Olfactory receptor 4A47 OR4A47 Q6IF82 Olfactory receptor 4C45 OR4C45 A6NMZ5 Olfactory receptor 4C46 OR4C46 A6NHA9 Olfactory receptor 4F15 OR4F15 Q8NGB8 Olfactory receptor 4F17 OR4F17 Q8NGA8 Olfactory receptor 4F21 OR4F21 O95013 Olfactory receptor 51A2 OR51A2 Q8NGJ7 Olfactory receptor 51A4 OR51A4 Q8NGJ6 Olfactory receptor 51A7 OR51A7 Q8NH64 Olfactory receptor 51B2 OR51B2 Q9Y5P1 Olfactory receptor 51B4 OR51B4 Q9Y5P0 Olfactory receptor 51B5 OR51B5 Q9H339 Olfactory receptor 51B5 OR51B6 Q9H340 Olfactory receptor 51D1 OR51D1 Q8NGF3 Olfactory receptor 51E1 OR51E1 Q8TCB6 Olfactory receptor 51E2 OR51E2 Q9H255 Olfactory receptor 51F1 OR51F1 A6NGY5 Olfactory receptor 51F2 OR51F2 Q8NH61 Olfactory receptor 51G1 OR51G1 Q8NGK1 Olfactory receptor 51G2 OR51G2 Q8NGK0 Putative olfactory receptor 51H1 OR51H1P Q8NH63 Olfactory receptor 51I1 OR51I1 Q9H343 Olfactory receptor 56A1 OR56A1 Q8NGH5 Olfactory receptor 56A3 OR56A3 Q8NH54 Olfactory receptor 56A4 OR56A4 Q8NGH8 Olfactory receptor 56A5 OR56A5 P0C7T3 Olfactory receptor 56B1 OR56B1 Q8NGI3 Olfactory receptor 56B4 OR56B4 Q8NH76 Olfactory receptor 5AC2 OR5AC2 Q9NZP5 Olfactory receptor 5AK2 OR5AK2 Q8NH90 Olfactory receptor 5AN1 OR5AN1 Q8NGI8 Olfactory receptor 5AP2 OR5AP2 Q8NGF4 Olfactory receptor 5AR1 OR5AR1 Q8NGP9 Olfactory receptor 5AS1 OR5AS1 Q8N127 Olfactory receptor 5AU1 OR5AU1 Q8NGC0 Olfactory receptor 5H14 OR5H14 A6NHG9 Olfactory receptor 5H15 OR5H15 A6NDH6 Olfactory receptor 6C65 OR6C65 A6NJZ3 Olfactory receptor 6C68 OR6C68 A6NDL8 Olfactory receptor 6C70 OR6C70 A6NIJ9 Olfactory receptor 6C74 OR6C74 A6NCV1 Olfactory receptor 6C75 OR6C75 A6NL08 Olfactory receptor 6C76 OR6C76 A6NM76 Olfactory receptor 7E24 OR7E24 Q6IFN5 Opsin-3 OPN3 Q9H1Y3 Melanopsin OPN4 Q9UHM6 Opsin-5 OPN5 Q6U736 δ-type opioid receptor OPRD1 P41143 κ-type opioid receptor OPRK1 P41145 μ-type opioid receptor OPRM1 P35372 Nociceptin receptor OPRL1 P41146 Blue-sensitive opsin OPN1SW P03999 Rhodopsin RHO P08100 Green-sensitive opsin OPN1MW P04001 Olfactory receptor 2B11 OR2B11 Q5JQS5 Olfactory receptor 2C1 OR2C1 O95371 Olfactory receptor 2C3 OR2C3 Q8N628 Olfactory receptor 2D2 OR2D2 Q9H210 Olfactory receptor 2D3 OR2D3 Q8NGH3 Olfactory receptor 2F1 OR2F1 Q13607 Olfactory receptor 2F2 OR2F2 O95006 Olfactory receptor 2G2 OR2G2 Q8NGZ5 Olfactory receptor 2G3 OR2G3 Q8NGZ4 Olfactory receptor 2G6 OR2G6 Q5TZ20 Olfactory receptor 2H1 OR2H1 Q9GZK4 Olfactory receptor 2H2 OR2H2 O95918 Putative olfactory receptor 2I1 OR2I1P Q8NGU4 Olfactory receptor 2J1 OR2J1 Q9GZK6 Olfactory receptor 2J2 OR2J2 O76002 Olfactory receptor 2J3 OR2J3 O76001 Olfactory receptor 2K2 OR2K2 Q8NGT1 Olfactory receptor 2L2 OR2L2 Q8NH16 Olfactory receptor 2L3 OR2L3 Q8NG85 Olfactory receptor 2L5 OR2L5 Q8NG80 Olfactory receptor 2L8 OR2L8 Q8NGY9 Olfactory receptor 2L13 OR2L13 Q8N349 Olfactory receptor 2M2 OR2M2 Q96R28 Olfactory receptor 2M3 OR2M3 Q8NG83 Olfactory receptor 2M4 OR2M4 Q96R27 Olfactory receptor 2M5 OR2M5 A3KFT3 Olfactory receptor 2M7 OR2M7 Q8NG81 Olfactory receptor 2S2 OR2S2 Q9NQN1 Olfactory receptor 2T1 OR2T1 O43869 Olfactory receptor 2T2 OR2T2 Q6IF00 Olfactory receptor 2T3 OR2T3 Q8NH03 Olfactory receptor 2T4 OR2T4 Q8NH00 Olfactory receptor 4E1 OR4E1 P0C645 Olfactory receptor 4E2 OR4E2 Q8NGC2 Olfactory receptor 4F3/4F16/4F29 OR4F3 Q6IEY1 Olfactory receptor 4F4 OR4F4 Q96R69 Olfactory receptor 4F5 OR4F5 Q8NH21 Olfactory receptor 4F6 OR4F6 Q8NGB9 Olfactory receptor 4K1 OR4K1 Q8NGD4 Olfactory receptor 4K2 OR4K2 Q8NGD2 Olfactory receptor 4K3 OR4K3 Q96R72 Olfactory receptor 4K5 OR4K5 Q8NGD3 Olfactory receptor 4K13 OR4K13 Q8NH42 Olfactory receptor 4K14 OR4K14 Q8NGD5 Olfactory receptor 4K15 OR4K15 Q8NH41 Olfactory receptor 4K17 OR4K17 Q8NGC6 Olfactory receptor 4L1 OR4L1 Q8NH43 Olfactory receptor 4M1 OR4M1 Q8NGD0 Olfactory receptor 4M2 OR4M2 Q8NGB6 Olfactory receptor 4N2 OR4N2 Q8NGD1 Olfactory receptor 4N4 OR4N4 Q8N0Y3 Olfactory receptor 4N5 OR4N5 Q8IXE1 Olfactory receptor 4P4 OR4P4 Q8NGL7 Olfactory receptor 4Q2 OR4Q2 P0C623 Olfactory receptor 4Q3 OR4Q3 Q8NH05 Olfactory receptor 4S1 OR4S1 Q8NGB4 Olfactory receptor 4S2 OR4S2 Q8NH73 Olfactory receptor 4X1 OR4X1 Q8NH49 Olfactory receptor 4X2 OR4X2 Q8NGF9 Olfactory receptor 5A1 OR5A1 Q8NGJ0 Olfactory receptor 5A2 OR5A2 Q8NGI9 Olfactory receptor 5B2 OR5B2 Q96R09 Olfactory receptor 5B3 OR5B3 Q8NH48 Olfactory receptor 5B12 OR5B12 Q96R08 Olfactory receptor 6A2 OR6A2 O95222 Olfactory receptor 6B1 OR6B1 O95007 Olfactory receptor 6B2 OR6B2 Q6IFH4 Olfactory receptor 6B3 OR6B3 Q8NGW1 Olfactory receptor 6C1 OR6C1 Q96RD1 Olfactory receptor 6C2 OR6C2 Q9NZP2 Olfactory receptor 6C3 OR6C3 Q9NZP0 Olfactory receptor 6C4 OR6C4 Q8NGE1 Olfactory receptor 6C6 OR6C6 A6NF89 Olfactory receptor 6F1 OR6F1 Q8NGZ6 Olfactory receptor 6J1 OR6J1 Q8NGC5 Olfactory receptor 6K2 OR6K2 Q8NGY2 Olfactory receptor 6K3 OR6K3 Q8NGY3 Olfactory receptor 6K6 OR6K6 Q8NGW6 Olfactory receptor 6M1 OR6M1 Q8NGM8 Olfactory receptor 6N1 OR6N1 Q8NGY5 Olfactory receptor 6N2 OR6N2 Q8NGY6 Olfactory receptor 6P1 OR6P1 Q8NGX9 Olfactory receptor 6Q1 OR6Q1 Q8NGQ2 Olfactory receptor 6S1 OR6S1 Q8NH40 Olfactory receptor 6T1 OR6T1 Q8NGN1 Olfactory receptor 6V1 OR6V1 Q8N148 Olfactory receptor 6X1 OR6X1 Q8NH79 Olfactory receptor 6Y1 OR6Y1 Q8NGX8 Olfactory receptor 7A5 OR7A5 Q15622 Olfactory receptor 7A10 OR7A10 O76100 Olfactory receptor 7A17 OR7A17 O14581 Olfactory receptor 7C1 OR7C1 O76099 Olfactory receptor 7C2 OR7C2 O60412 Olfactory receptor 7D4 OR7D4 Q8NG98 Olfactory receptor 7G1 OR7G1 Q8NGA0 Olfactory receptor 7G2 OR7G2 Q8NG99 Prostaglandin F2-α receptor PTGFR P43088 Prostacyclin receptor PTGIR P43119 Prolactin-releasing peptide receptor PRLHR P49683 Platelet-activating factor receptor PTAFR P25105 Pyroglutamylated RFamide peptide receptor QRFPR Q96P65 RPE-retinal G protein-coupled receptor RGR P47804 Sphingosine 1-phosphate receptor 1 S1PR1 P21453 Sphingosine 1-phosphate receptor 2 S1PR2 O95136 Sphingosine 1-phosphate receptor 3 S1PR3 Q99500 Sphingosine 1-phosphate receptor 4 S1PR4 O95977 Sphingosine 1-phosphate receptor 5 S1PR5 Q9H228 Somatostatin receptor type 1 SSTR1 P30872 Somatostatin receptor type 2 SSTR2 P30874 Somatostatin receptor type 3 SSTR3 P32745 Somatostatin receptor type 4 SSTR4 P31391 Somatostatin receptor type 5 SSTR5 P35346 Thromboxane A2 receptor TBXA2R P21731 Trace amine-associated receptor 1 TAAR1 Q96RJ0 Trace amine-associated receptor 2 TAAR2 Q9P1P5 Putative trace amine-associated receptor 3 TAAR3 Q9P1P4 Trace amine-associated receptor 5 TAAR5 O14804 Trace amine-associated receptor 6 TAAR6 Q96RI8 Trace amine-associated receptor 8 TAAR8 Q969N4 Trace amine-associated receptor 9 TAAR9 Q96RI9 Thyrotropin receptor TSHR P16473 Vasopressin V1a receptor AVPR1A P37288 Vasopressin V1b receptor AVPR1B P47901 Vasopressin V2 receptor AVPR2 P30518 Chemokine XC receptor 1 XCR1 P46094 Brain-specific angiogenesis inhibitor 1 BAI1 O14514 Brain-specific angiogenesis inhibitor 2 BAI2 O60241 Brain-specific angiogenesis inhibitor 3 BAI3 O60242 Calcitonin receptor CALCR P30988 Calcitonin gene-related peptide type 1 CALCRL Q16602 receptor Corticotropin-releasing factor receptor 1 CRHR1 P34998 Corticotropin-releasing factor receptor 2 CRHR2 Q13324 Growth hormone-releasing hormone receptor GHRHR Q02643 Gastric inhibitory polypeptide receptor GIPR P48546 Glucagon-like peptide 1 receptor GLP1R P43220 Glucagon-like peptide 2 receptor GLP2R O95838 Glucagon receptor GCGR P47871 Pituitary adenylate cyclase-activating ADCYAP1R1 P41586 polypeptide type I receptor Taste receptor 1 member 2 TAS1R2 Q8TE23 Parathyroid hormone receptor 1 PTH1R Q03431 Parathyroid hormone 2 receptor PTH2R P49190 Secretin receptor SCTR P47872 Vasoactive intestinal polypeptide receptor 1 VIPR1 P32241 Vasoactive intestinal polypeptide receptor 2 VIPR2 P41587 Frizzled-10 FZD10 Q9ULW2 Frizzled-1 FZD1 Q9UP38 Frizzled-2 FZD2 Q14332 Frizzled-3 FZD3 Q9NPG1 Frizzled-4 FZD4 Q9ULV1 Frizzled-5 FZD5 Q13467 Frizzled-6 FZD6 O60353 Frizzled-7 FZD7 O75084 Frizzled-8 FZD8 Q9H461 Frizzled-9 FZD9 (FZD3) O00144 Smoothened homologue SMO (SMOH) Q99835 Extracellular calcium-sensing receptor CASR P41180 GABA type B receptor 1subunit 1 GABBR1 Q9UBS5 GABA type B receptor subunit 2 GABBR2 O75899 GPCR family C group 6 member A GPRC6A Q5T6X5 Metabotropic glutamate receptor 1 GRM1 Q13255 Metabotropic glutamate receptor 2 GRM2 Q14416 Metabotropic glutamate receptor 3 GRM3 Q14832 Metabotropic glutamate receptor 4 GRM4 Q14833 Metabotropic glutamate receptor 5 GRM5 P41594 Metabotropic glutamate receptor 6 GRM6 O15303 Metabotropic glutamate receptor 7 GRM7 Q14831 Metabotropic glutamate receptor 8 GRM8 O00222 Taste receptor 1 member 1 TAS1R1 Q7RTX1 Taste receptor 1 member 2 TAS1R2 Q8TE23 Taste receptor 1 member 3 TAS1R3 Q7RTX0

Described herein are GPCR binding domains, wherein the GPCR binding domains are designed based on surface interactions between a GPCR ligand and the GPCR. In some instances, the ligand is a subatomic particle (e.g., a photon), an ion, an organic molecule, a peptide, and a protein. Non-limiting examples of ligands which can be bound by a GPCR include (−)-adrenaline, (−)-noradrenaline, (lyso)phospholipid mediators, [des-Arg10]kallidin, [des-Arg9]bradykinin, [des-Gln14]ghrelin, [Hyp3]bradykinin, [Leu]enkephalin, [Met]enkephalin, 12-hydroxyheptadecatrienoic acid, 12R-HETE, 12S-HETE, 12S-HPETE, 15S-HETE, 17β-estradiol, 20-hydroxy-LTB4, 2-arachidonoylglycerol, 2-oleoyl-LPA, 3-hydroxyoctanoic acid, 5-hydroxytryptamine, 5-oxo-15-HETE, 5-oxo-ETE, 5-oxo-ETrE, 5-oxo-ODE, 5S-HETE, 5S-HPETE, 7α,25-dihydroxycholesterol, acetylcholine, ACTH, adenosine diphosphate, adenosine, adrenomedullin 2/intermedin, adrenomedullin, amylin, anandamide, angiotensin II, angiotensin III, annexin I, apelin receptor early endogenous ligand, apelin-13, apelin-17, apelin-36, aspirin triggered lipoxin A4, aspirin-triggered resolvin D1, ATP, beta-defensin 4A, big dynorphin, bovine adrenal medulla peptide 8-22, bradykinin, C3a, C5a, Ca2+, calcitonin gene related peptide, calcitonin, cathepsin G, CCK-33, CCK-4, CCK-8, CCL1, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL7, CCL8, chemerin, chenodeoxycholic acid, cholic acid, corticotrophin-releasing hormone, CST-17, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12α, CXCL12β, CXCL13, CXCL16, CXCL2, CXCL3, CXCLS, CXCL6, CXCL7, CXCL8, CXCL9, cysteinyl-leukotrienes (CysLTs), uracil nucleotides, deoxycholic acid, dihydrosphingosine-1-phosphate, dioleoylphosphatidic acid, dopamine, dynorphin A, dynorphin A-(1-13), dynorphin A-(1-8), dynorphin B, endomorphin-1, endothelin-1, endothelin-2, endothelin-3, F2L, Free fatty acids, FSH, GABA, galanin, galanin-like peptide, gastric inhibitory polypeptide, gastrin-17, gastrin-releasing peptide, ghrelin, GHRH, glucagon, glucagon-like peptide 1-(7-36) amide, glucagon-like peptide 1-(7-37), glucagon-like peptide 2, glucagon-like peptide 2-(3-33), GnRH I, GnRH II, GRP-(18-27), hCG, histamine, humanin, INSL3, INSL5, kallidin, kisspeptin-10, kisspeptin-13, kisspeptin-14, kisspeptin-54, kynurenic acid, large neuromedin N, large neurotensin, L-glutamic acid, LH, lithocholic acid, L-lactic acid, long chain carboxylic acids, LPA, LTB4, LTC4, LTD4, LTE4, LXA4, Lys-[Hyp3]-bradykinin, lysophosphatidylinositol, lysophosphatidylserine, Medium-chain-length fatty acids, melanin-concentrating hormone, melatonin, methylcarbamyl PAF, Mg2+, motilin, N-arachidonoylglycine, neurokinin A, neurokinin B, neuromedin B, neuromedin N, neuromedin S-33, neuromedin U-25, neuronostatin, neuropeptide AF, neuropeptide B-23, neuropeptide B-29, neuropeptide FF, neuropeptide S, neuropeptide SF, neuropeptide W-23, neuropeptide W-30, neuropeptide Y, neuropeptide Y-(3-36), neurotensin, nociceptin/orphanin FQ, N-oleoylethanolamide, obestatin, octopamine, orexin-A, orexin-B, Oxysterols, oxytocin, PACAP-27, PACAP-38, PAF, pancreatic polypeptide, peptide YY, PGD2, PGE2, PGF2a, PGI2, PGJ2, PHM, phosphatidylserine, PHV, prokineticin-1, prokineticin-2, prokineticin-20, prosaposin, PrRP-20, PrRP-31, PTH, PTHrP, PTHrP-(1-36), QRFP43, relaxin, relaxin-1, relaxin-3, resolvin D1, resolvin E1, RFRP-1, RFRP-3, R-spondins, secretin, serine proteases, sphingosine 1-phosphate, sphingosylphosphorylcholine, SRIF-14, SRIF-28, substance P, succinic acid, thrombin, thromboxane A2, TIP39, T-kinin, TRH, TSH, tyramine, UDP-glucose, uridine diphosphate, urocortin 1, urocortin 2, urocortin 3, urotensin II-related peptide, urotensin-II, vasopressin, VIP, Wnt, Wnt-1, Wnt-10a, Wnt-10b, Wnt-11, Wnt-16, Wnt-2, Wnt-2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt-9a, Wnt-9b, XCL1, XCL2, Zn2+, α-CGRP, α-ketoglutaric acid, α-MSH, α-neoendorphin, β-alanine, β-CGRP, β-D-hydroxybutyric acid, β-endorphin, β-MSH, β-neoendorphin, β-phenylethylamine, and γ-MSH.

Sequences of GPCR binding domains based on surface interactions between a GPCR ligand and the GPCR are analyzed using various methods. For example, multispecies computational analysis is performed. In some instances, a structure analysis is performed. In some instances, a sequence analysis is performed. Sequence analysis can be performed using a database known in the art. Non-limiting examples of databases include, but are not limited to, NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), UCSC Genome Browser (genome.ucsc.edu/), UniProt (www.uniprot.org/), and IUPHAR/BPS Guide to PHARMACOLOGY (guidetopharmacology.org/).

Described herein are GPCR binding domains designed based on sequence analysis among various organisms. For example, sequence analysis is performed to identify homologous sequences in different organisms. Exemplary organisms include, but are not limited to, mouse, rat, equine, sheep, cow, primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly, and human.

Following identification of GPCR binding domains, libraries comprising nucleic acids encoding for the GPCR binding domains may be generated. In some instances, libraries of GPCR binding domains comprise sequences of GPCR binding domains designed based on conformational ligand interactions, peptide ligand interactions, small molecule ligand interactions, extracellular domains of GPCRs, or antibodies that target GPCRs. Libraries of GPCR binding domains may be translated to generate protein libraries. In some instances, libraries of GPCR binding domains are translated to generate peptide libraries, immunoglobulin libraries, derivatives thereof, or combinations thereof. In some instances, libraries of GPCR binding domains are translated to generate protein libraries that are further modified to generate peptidomimetic libraries. In some instances, libraries of GPCR binding domains are translated to generate protein libraries that are used to generate small molecules.

Methods described herein provide for synthesis of libraries of GPCR binding domains comprising nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the libraries of GPCR binding domains comprise varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GPCR binding domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GPCR binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of libraries comprising nucleic acids encoding for the GPCR binding domains, wherein the libraries comprise sequences encoding for variation of length of the GPCR binding domains. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Following identification of GPCR binding domains, the GPCR binding domains may be placed in scaffolds as described herein. In some instances, the scaffolds are immunoglobulins. In some instances, the GPCR binding domains are placed in the CDR-H3 region. GPCR binding domains that may be placed in scaffolds can also be referred to as a motif. Scaffolds comprising GPCR binding domains may be designed based on binding, specificity, stability, expression, folding, or downstream activity. In some instances, the scaffolds comprising GPCR binding domains enable contact with the GPCRs. In some instances, the scaffolds comprising GPCR binding domains enables high affinity binding with the GPCRs. Exemplary amino acid sequences of GPCR binding domains are described in Table 2.

TABLE 2 GPCR amino acid sequences SEQ ID NO GPCR Amino Acid Sequence 1 CXCR4 MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFL TGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVD AVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQ RPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYPND LWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVI LILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFH CCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTE SESSSFHSS 2 CCR4 MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSLVF VFGLLGNSVVVLVLFKYKRLRSMTDVYLLNLAISDLLFVFSLPFWGY YAADQWVFGLGLCKMISWMYLVGFYSGIFFVMLMSIDRYLAIVHAV FSLRARTLTYGVITSLATWSVAVFASLPGFLFSTCYTERNHTYCKTKY SLNSTTWKVLSSLEINILGLVIPLGIMLFCYSMIIRTLQHCKNEKKNKA VKMIFAVVVLFLGFWTPYNIVLFLETLVELEVLQDCTFERYLDYAIQA TETLAFVHCCLNPIIYFFLGEKFRKYILQLFKTCRGLFVLCQYCGLLQI YSADTPSSSYTQSTMDHDLHDAL 3 GCGR MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHN LSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQH RFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMY SSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVL KASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQ YGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFV VPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQ LLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQ GTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLG KVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGG LPRLAESPF 4 mGluR5 MVLLLILSVLLLKEDVRGSAQSSERRVVAHMPGDIIIGALFSVHHQPT VDKVHERKCGAVREQYGIQRVEAMLHTLERINSDPTLLPNITLGCEIR DSCWHSAVALEQSIEFIRDSLISSEEEEGLVRCVDGSSSSFRSKKPIVGV IGPGSSSVAIQVQNLLQLFNIPQIAYSATSMDLSDKTLFKYFMRVVPSD AQQARAMVDIVKRYNWTYVSAVHTEGNYGESGMEAFKDMSAKEGI CIAHSYKIYSNAGEQSFDKLLKKLTSHLPKARVVACFCEGMTVRGLL MAMRRLGLAGEFLLLGSDGWADRYDVTDGYQREAVGGITIKLQSPD VKWFDDYYLKLRPETNHRNPWFQEFWQHRFQCRLEGFPQENSKYNK TCNSSLTLKTHHVQDSKMGFVINAIYSMAYGLHNMQMSLCPGYAGL CDAMKPIDGRKLLESLMKTNFTGVSGDTILFDENGDSPGRYEIMNFKE MGKDYFDYINVGSWDNGELKMDDDEVWSKKSNIIRSVCSEPCEKGQI KVIRKGEVSCCWTCTPCKENEYVFDEYTCKACQLGSWPTDDLTGCD LIPVQYLRWGDPEPIAAVVFACLGLLATLFVTVVFIIYRDTPVVKSSSR ELCYIILAGICLGYLCTFCLIAKPKQIYCYLQRIGIGLSPAMSYSALVTK TNRIARILAGSKKKICTKKPRFMSACAQLVIAFILICIQLGIIVALFIMEP PDIMHDYPSIREVYLICNTTNLGVVTPLGYNGLLILSCTFYAFKTRNVP ANFNEAKYIAFTMYTTCIIWLAFVPIYFGSNYKIITMCFSVSLSATVAL GCMFVPKVYIILAKPERNVRSAFTTSTVVRMHVGDGKSSSAASRSSSL VNLWKRRGSSGETLRYKDRRLAQHKSEIECFTPKGSMGNGGRATMS SSNGKSVTWAQNEKSSRGQHLWQRLSIHINKKENPNQTAVIKPFPKST ESRGLGAGAGAGGSAGGVGATGGAGCAGAGPGGPESPDAGPKALY DVAEAEEHFPAPARPRSPSPISTLSHRAGSASRTDDDVPSLHSEPVARS SSSQGSLMEQISSVVTRFTANISELNSMMLSTAAPSPGVGAPLCSSYLI PKEIQLPTTMTTFAEIQPLPAIEVTGGAQPAAGAQAAGDAARESPAAG PEAAAAKPDLEELVALTPPSPFRDSVDSGSTTPNSPVSESALCIPSSPKY DTLIIRDYTQSSSSL 5 GLP-1R RPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYA CWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDNS SLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIASAIL LGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQ WDGLLSYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSV LSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNY WLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKCRLAKSTLT LIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCF VNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSLSSGATAGS SMYTATCQASCS 6 GABAB MLLLLLLAPLFLRPPGAGGAQTPNATSEGCQIIHPPWEGGIRYRGLTR DQVKAINFLPVDYEIEYVCRGEREVVGPKVRKCLANGSWTDMDTPS RCVRICSKSYLTLENGKVFLTGGDLPALDGARVDFRCDPDFHLVGSS RSICSQGQWSTPKPHCQVNRTPHSERRAVYIGALFPMSGGWPGGQAC QPAVEMALEDVNSRRDILPDYELKLIHHDSKCDPGQATKYLYELLYN DPIKIILMPGCSSVSTLVAEAARMWNLIVLSYGSSSPALSNRQRFPTFF RTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSTLDDLEERVKE AGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKE RLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIV MLNPANTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIW ALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVS GHVVFDASGSRMAWTLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKW IGGSPPADQTLVIKTFRFLSQKLFISVSVLSSLGIVLAVVCLSFNIYNSH VRYIQNSQPNLNNLTAVGCSLALAAVFPLGLDGYHIGRNQFPFVCQA RLWLLGLGFSLGYGSMFTKIWWVHTVFTKKEEKKEWRKTLEPWKL YATVGLLVGMDVLTLAIWQIVDPLHRTIETFAKEEPKEDIDVSILPQLE HCSSRKMNTWLGIFYGYKGLLLLLGIFLAYETKSVSTEKINDHRAVG MAIYNVAVLCLITAPVTMILSSQQDAAFAFASLAIVFSSYITLVVLFVP KMRRLITRGEWQSEAQDTMKTGSSTNNNEEEKSRLLEKENRELEKIIA EKEERVSELRHQLQSRQQLRSRRHPPTPPEPSGGLPRGPPEPPDRLSCD GSRVHLLYK 7 OPRM1 MDSSAAPTNASNCTDALAYSSCSPAPSPGSWVNLSHLDGNLSDPCGP NRTDLGGRDSLCPPTGSPSMITAITIMALYSIVCVVGLFGNFLVMYVIV RYTKMKTATNIYIFNLALADALATSTLPFQSVNYLMGTWPFGTILCKI VISIDYYNMFTSIFTLCTMSVDRYIAVCHPVKALDFRTPRNAKIINVCN WILSSAIGLPVMFMATTKYRQGSIDCTLTFSHPTWYWENLLKICVFIF AFIMPVLIITVCYGLMILRLKSVRMLSGSKEKDRNLRRITRMVLVVVA VFIVCWTPIHIYVIIKALVTIPETTFQTVSWHFCIALGYTNSCLNPVLYA FLDENFKRCFREFCIPTSSNIEQQNSTRIRQNTRDHPSTANTVDRTNHQ LENLEAETAPLP 8 OPRK1 MDSPIQIFRGEPGPTCAPSACLPPNSSAWFPGWAEPDSNGSAGSEDAQ LEPAHISPAIPVIITAVYSVVFVVGLVGNSLVMFVIIRYTKMKTATNIYI FNLALADALVTTTMPFQSTVYLMNSWPFGDVLCKIVISIDYYNMFTSI FTLTMMSVDRYIAVCHPVKALDFRTPLKAKIINICIWLLSSSVGISAIVL GGTKVREDVDVIECSLQFPDDDYSWWDLFMKICVFIFAFVIPVLIIIVC YTLMILRLKSVRLLSGSREKDRNLRRITRLVLVVVAVFVVCWTPIHIFI LVEALGSTSHSTAALSSYYFCIALGYTNSSLNPILYAFLDENFKRCFRD FCFPLKMRMERQSTSRVRNTVQDPAYLRDIDGMNKPV 9 C5aR MDSFNYTTPDYGHYDDKDTLDLNTPVDKTSNTLRVPDILALVIFAVV FLVGVLGNALVVWVTAFEAKRTINAIWFLNLAVADFLSCLALPILFTS IVQHHHWPFGGAACSILPSLILLNMYASILLLATISADRFLLVFKPIWC QNFRGAGLAWIACAVAWGLALLLTIPSFLYRVVREEYFPPKVLCGVD YSHDKRRERAVAIVRLVLGFLWPLLTLTICYTFILLRTWSRRATRSTK TLKVVVAVVASFFIFWLPYQVTGIMMSFLEPSSPTFLLLKKLDSLCVSF AYINCCINPIIYVVAGQGFQGRLRKSLPSLLRNVLTEESVVRESKSFTR STVDTMAQKTQAV 10 CGRP ELEESPEDSIQLGVTRNKIMTAQYECYQKIMQDPIQQAEGVYCNRTW DGWLCWNDVAAGTESMQLCPDYFQDFDPSEKVTKICDQDGNWFRH PASNRTWTNYTQCNVNTHEKVKTALNLFYLTIIGHGLSIASLLISLGIF FYFKSLSCQRITLHKNLFFSFVCNSVVTIIHLTAVANNQALVATNPVSC KVSQFIHLYLMGCNYFWMLCEGIYLHTLIVVAVFAEKQHLMWYYFL GWGFPLIPACIHAIARSLYYNDNCWISSDTHLLYIIHGPICAALLVNLFF LLNIVRVLITKLKVTHQAESNLYMKAVRATLILVPLLGIEFVLIPWRPE GKIAEEVYDYIMHILMHFQGLLVSTIFCFFNGEVQAILRRNWNQYKIQ FGNSFSNSEALRSASYTVSTISDGPGYSHDCPSEHLNGKSIHDIENVLL KPENLYN 11 M1 MNTSAPPAVSPNITVLAPGKGPWQVAFIGITTGLLSLATVTGNLLVLIS muscarinic FKVNTELKTVNNYFLLSLACADLIIGTFSMNLYTTYLLMGHWALGTL ACDLWLALDYVASNASVMNLLLISFDRYFSVTRPLSYRAKRTPRRAA LMIGLAWLVSFVLWAPAILFWQYLVGERTVLAGQCYIQFLSQPIITFG TAMAAFYLPVTVMCTLYWRIYRETENRARELAALQGSETPGKGGGS SSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSM ESLTSSEGEEPGSEVVIKMPMVDPEAQAPTKQPPRSSPNTVKRPTKKG RDRAGKGQKPRGKEQLAKRKTFSLVKEKKAARTLSAILLAFILTWTP YNIMVLVSTFCKDCVPETLWELGYWLCYVNSTINPMCYALCNKAFR DTFRLLLLCRWDKRRWRKIPKRPGSVHRTPSRQC 12 M4 MANFTPVNGSSGNQSVRLVTSSSHNRYETVEMVFIATVTGSLSLVTV muscarinic VGNILVMLSIKVNRQLQTVNNYFLFSLACADLIIGAFSMNLYTVYIIKG YWPLGAVVCDLWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPAR RTTKMAGLMIAAAWVLSFVLWAPAILFWQFVVGKRTVPDNQCFIQF LSNPAVTFGTAIAAFYLPVVIMTVLYIHISLASRSRVHKHRPEGPKEKK AKTLAFLKSPLMKQSVKKPPPGEAAREELRNGKLEEAPPPALPPPPRP VADKDTSNESSSGSATQNTKERPATELSTTEATTPAMPAPPLQPRALN PASRWSKIQIVTKQTGNECVTAIEIVPATPAGMRPAANVARKFASIAR NQVRKKRQMAARERKVTRTIFAILLAFILTWTPYNVMVLVNTFCQSC IPDTVWSIGYWLCYVNSTINPACYALCNATFKKTFRHLLLCQYRNIGT AR 13 CCR2 MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPL YSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLW AHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVF ALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKEDSVYVCGPYFP RGWNNFHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRAVR VIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTETL GMTHCCINPIIYAFVGEKFRSLFHIALGCRIAPLQKPVCGGPGVRPGKN VKVTTQGLLDGRGKGKSIGRAPEASLQDKEGA 14 CCR9 MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNVRQFA SHFLPPLYWLVFIVGALGNSLVILVYWYCTRVKTMTDMFLLNLAIAD LLFLVTLPFWAIAAADQWKFQTFMCKVVNSMYKMNFYSCVLLIMCI SVDRYIAIAQAMRAHTWREKRLLYSKMVCFTIWVLAAALCIPEILYS QIKEESGIAICTMVYPSDESTKLKSAVLTLKVILGFFLPFVVMACCYTII IHTLIQAKKSSKHKALKVTITVLTVFVLSQFPYNCILLVQTIDAYAMFI SNCAVSTNIDICFQVTQTIAFFHSCLNPVLYVFVGERFRRDLVKTLKNL GCISQAQWVSFTRREGSLKLSSMLLETTSGALSL 15 GPR174 MPANYTCTRPDGDNTDFRYFIYAVTYTVILVPGLIGNILALWVFYGY MKETKRAVIFMINLAIADLLQVLSLPLRIFYYLNHDWPFGPGLCMFCF YLKYVNMYASIYFLVCISVRRFWFLMYPFRFHDCKQKYDLYISIAGW LIICLACVLFPLLRTSDDTSGNRTKCFVDLPTRNVNLAQSVVMMTIGE LIGFVTPLLIVLYCTWKTVLSLQDKYPMAQDLGEKQKALKMILTCAG VFLICFAPYHFSFPLDFLVKSNEIKSCLARRVILIFHSVALCLASLNSCL DPVIYYFSTNEFRRRLSRQDLHDSIQLHAKSFVSNHTASTMTPELC 16 MASP-2 TPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFT HFDLELSHLCEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSL GSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCH NHLGGFYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRP YPKLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYDFLKIQTDREE HGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYP MAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKD GSWDRPMPACSIVDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEET FYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVCGLSARTTGGRI YGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKH DASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKL NNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQRGFLARNLM YVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDS GGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIE NIISDF 17 CCR5 MDYQVSSPIYDINYYTSEPCQKINVKQIAARLLPPLYSLVFIFGFVGNM LVILILINCKRLKSMTDIYLLNLAISDLFFLLTVPFWAHYAAAQWDFG NTMCQLLTGLYFIGFFSGIFFIILLTIDRYLAVVHAVFALKARTVTFGV VTSVITWVVAVFASLPGIIFTRSQKEGLHYTCSSHFPYSQYQFWKNFQ TLKIVILGLVLPLLVMVICYSGILKTLLRCRNEKKRHRAVRLIFTIMIVY FLFWAPYNIVLLLNTFQEFFGLNNCSSSNRLDQAMQVTETLGMTHCCI NPIIYAFVGEKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTR STGEQEISVGL 18 FSHR CHHRICHCSNRVFLCQESKVTEIPSDLPRNAIELRFVLTKLRVIQKGAF SGFGDLEKIEISQNDVLEVIEADVFSNLPKLHEIRIEKANNLLYINPEAF QNLPNLQYLLISNTGIKHLPDVHKIHSLQKVLLDIQDNINIHTIERNSFV GLSFESVILWLNKNGIQEIHNCAFNGTQLDELNLSDNNNLEELPNDVF HGASGPVILDISRTRIHSLPSYGLENLKKLRARSTYNLKKLPTLEKLVA LMEASLTYPSHCCAFANWRRQISELHPICNKSILRQEVDYMTQARGQ RSSLAEDNESSYSRGFDMTYTEFDYDLCNEVVDVTCSPKPDAFNPCE DIMGYNILRVLIWFISILAITGNIIVLVILTTSQYKLTVPRFLMCNLAFA DLCIGIYLLLIASVDIHTKSQYHNYAIDWQTGAGCDAAGFFTVFASEL SVYTLTAITLERWHTITHAMQLDCKVQLRHAASVMVMGWIFAFAAA LFPIFGISSYMKVSICLPMDIDSPLSQLYVMSLLVLNVLAFVVICGCYIH IYLTVRNPNIVSSSSDTRIAKRMAMLIFTDFLCMAPISFFAISASLKVPLI TVSKAKILLVLFHPINSCANPFLYAIFTKNFRRDFFILLSKCGCYEMQA QIYRTETSSTVHNTHPRNGHCSSAPRVTNGSTYILVPLSHLAQN 19 mGluR2 EGPAKKVLTLEGDLVLGGLFPVHQKGGPAEDCGPVNEHRGIQRLEA PAM MLFALDRINRDPHLLPGVRLGAHILDSCSKDTHALEQALDFVRASLSR GADGSRHICPDGSYATHGDAPTAITGVIGGSYSDVSIQVANLLRLFQIP QISYASTSAKLSDKSRYDYFARTVPPDFFQAKAMAEILRFFNWTYVST VASEGDYGETGIEAFELEARARNICVATSEKVGRAMSRAAFEGVVRA LLQKPSARVAVLFTRSEDARELLAASQRLNASFTWVASDGWGALES VVAGSEGAAEGAITIELASYPISDFASYFQSLDPWNNSRNPWFREFWE QRFRCSFRQRDCAAHSLRAVPFEQESKIMFVVNAVYAMAHALHNMH RALCPNTTRLCDAMRPVNGRRLYKDFVLNVKFDAPFRPADTHNEVR FDRFGDGIGRYNIFTYLRAGSGRYRYQKVGYWAEGLTLDTSLIPWAS PSAGPLPASRCSEPCLQNEVKSVQPGEVCCWLCIPCQPYEYRLDEFTC ADCGLGYWPNASLTGCFELPQEYIRWGDAWAVGPVTIACLGALATL FVLGVFVRHNATPVVKASGRELCYILLGGVFLCYCMTFIFIAKPSTAV CTLRRLGLGTAFSVCYSALLTKTNRIARIFGGAREGAQRPRFISPASQV AICLALISGQLLIVVAWLVVEAPGTGKETAPERREVVTLRCNHRDAS MLGSLAYNVLLIALCTLYAFKTRKCPENFNEAKFIGFTMYTTCIIWLA FLPIFYVTSSDYRVQTTTMCVSVSLSGSVVLGCLFAPKLHIILFQPQKN VVSHRAPTSRFGSAAARASSSLGQGSGSQFVPTVCNGREVVDSTTSSL 20 mGluR3 LGDHNFLRREIKIEGDLVLGGLFPINEKGTGTEECGRINEDRGIQRLEA MLFAIDEINKDDYLLPGVKLGVHILDTCSRDTYALEQSLEFVRASLTK VDEAEYMCPDGSYAIQENIPLLIAGVIGGSYSSVSIQVANLLRLFQIPQI SYASTSAKLSDKSRYDYFARTVPPDFYQAKAMAEILRFFNWTYVSTV ASEGDYGETGIEAFEQEARLRNICIATAEKVGRSNIRKSYDSVIRELLQ KPNARVVVLFMRSDDSRELIAAASRANASFTWVASDGWGAQESIIKG SEHVAYGAITLELASQPVRQFDRYFQSLNPYNNHRNPWFRDFWEQKF QCSLQNKRNHRRVCDKHLAIDSSNYEQESKIMFVVNAVYAMAHALH KMQRTLCPNTTKLCDAMKILDGKKLYKDYLLKINFTAPFNPNKDADS IVKFDTFGDGMGRYNVFNFQNVGGKYSYLKVGHWAETLSLDVNSIH WSRNSVPTSQCSDPCAPNEMKNMQPGDVCCWICIPCEPYEYLADEFT CMDCGSGQWPTADLTGCYDLPEDYIRWEDAWAIGPVTIACLGFMCT CMVVTVFIKHNNTPLVKASGRELCYILLFGVGLSYCMTFFFIAKPSPVI CALRRLGLGSSFAICYSALLTKTNCIARIFDGVKNGAQRPKFISPSSQV FICLGLILVQIVMVSVWLILEAPGTRRYTLAEKRETVILKCNVKDSSM LISLTYDVILVILCTVYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLP IFYVTSSDYRVQTTTMCISVSLSGFVVLGCLFAPKVHIILFQPQKNVVT HRLHLNRFSVSGTGTTYSQSSASTYVPTVCNGREVLDSTTSSL 21 mGluR4 KPKGHPHMNSIRIDGDITLGGLFPVHGRGSEGKPCGELKKEKGIHRLE AMLFALDRINNDPDLLPNITLGARILDTCSRDTHALEQSLTFVQALIEK DGTEVRCGSGGPPIITKPERVVGVIGASGSSVSIMVANILRLFKIPQISY ASTAPDLSDNSRYDFFSRVVPSDTYQAQAMVDIVRALKWNYVSTVA SEGSYGESGVEAFIQKSREDGGVCIAQSVKIPREPKAGEFDKIIRRLLET SNARAVIIFANEDDIRRVLEAARRANQTGHFFWMGSDSWGSKIAPVL HLEEVAEGAVTILPKRMSVRGFDRYFSSRTLDNNRRNIWFAEFWEDN FHCKLSRHALKKGSHVKKCTNRERIGQDSAYEQEGKVQFVIDAVYA MGHALHAMHRDLCPGRVGLCPRMDPVDGTQLLKYIRNVNFSGIAGN PVTFNENGDAPGRYDIYQYQLRNDSAEYKVIGSWTDHLHLRIERMH WPGSGQQLPRSICSLPCQPGERKKTVKGMPCCWHCEPCTGYQYQVD RYTCKTCPYDMRPTENRTGCRPIPIIKLEWGSPWAVLPLFLAVVGIAA TLFVVITFVRYNDTPIVKASGRELSYVLLAGIFLCYATTFLMIAEPDLG TCSLRRIFLGLGMSISYAALLTKTNRIYRIFEQGKRSVSAPRFISPASQL AITFSLISLQLLGICVWFVVDPSHSVVDFQDQRTLDPRFARGVLKCDIS DLSLICLLGYSMLLMVTCTVYAIKTRGVPETFNEAKPIGFTMYTTCIV WLAFIPIFFGTSQSADKLYIQTTTLTVSVSLSASVSLGMLYMPKVYIILF HPEQNVPKRKRSLKAVVTAATMSNKFTQKGNFRPNGEAKSELCENL EAPALATKQTYVTYTNHAI 22 mGluR7 QEMYAPHSIRIEGDVTLGGLFPVHAKGPSGVPCGDIKRENGIHRLEAM LYALDQINSDPNLLPNVTLGARILDTCSRDTYALEQSLTFVQALIQKD TSDVRCTNGEPPVFVKPEKVVGVIGASGSSVSIMVANILRLFQIPQISY ASTAPELSDDRRYDFFSRVVPPDSFQAQAMVDIVKALGWNYVSTLAS EGSYGEKGVESFTQISKEAGGLCIAQSVRIPQERKDRTIDFDRIIKQLLD TPNSRAVVIFANDEDIKQILAAAKRADQVGHFLWVGSDSWGSKINPL HQHEDIAEGAITIQPKRATVEGFDAYFTSRTLENNRRNVWFAEYWEE NFNCKLTISGSKKEDTDRKCTGQERIGKDSNYEQEGKVQFVIDAVYA MAHALHHMNKDLCADYRGVCPEMEQAGGKKLLKYIRNVNFNGSAG TPVMFNKNGDAPGRYDIFQYQTTNTSNPGYRLIGQWTDELQLNIEDM QWGKGVREIPASVCTLPCKPGQRKKTQKGTPCCWTCEPCDGYQYQF DEMTCQHCPYDQRPNENRTGCQDIPIIKLEWHSPWAVIPVFLAMLGII ATIFVMATFIRYNDTPIVRASGRELSYVLLTGIFLCYIITFLMIAKPDVA VCSFRRVFLGLGMCISYAALLTKTNRIYRIFEQGKKSVTAPRLISPTSQ LAITSSLISVQLLGVFIWFGVDPPNIIIDYDEHKTMNPEQARGVLKCDIT DLQIICSLGYSILLMVTCTVYAIKTRGVPENFNEAKPIGFTMYTTCIVW LAFIPIFFGTAQSAEKLYIQTTTLTISMNLSASVALGMLYMPKVYIIIFH PELNVQKRKRSFKAVVTAATMSSRLSHKPSDRPNGEAKTELCENVDP NSPAAKKKYVSYNNLVI 23 CXCR3 MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDF SLNFDRAFLPALYSLLFLLGLLGNGAVAAVLLSRRTALSSTDTFLLHL AVADTLLVLTLPLWAVDAAVQWVFGSGLCKVAGALFNINFYAGALL LACISFDRYLNIVHATQLYRRGPPARVTLTCLAVWGLCLLFALPDFIFL SAHHDERLNATHCQYNFPQVGRTALRVLQLVAGFLLPLLVMAYCYA HILAVLLVSRGQRRLRAMRLVVVVVVAFALCWTPYHLVVLVDILMD LGALARNCGRESRVDVAKSVTSGLGYMHCCLNPLLYAFVGVKFRER MWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL 24 CCR8 MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVF SLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLD QWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALK VRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQ TLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRL VLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIIS FTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKS SSCQQHSSRSSSVDYIL 25 Adenosine MPIMGSSVYITVELAIAVLAILGNVLVCWAVWLNSNLQNVTNYFVVS A2a LAAADIAVGVLAIPFAITISTGFCAACHGCLFIACFVLVLTQSSIFSLLAI AIDRYIAIRIPLRYNGLVTGTRAKGIIAICWVLSFAIGLTPMLGWNNCG QPKEGKNHSQGCGEGQVACLFEDVVPMNYMVYFNFFACVLVPLLL MLGVYLRIFLAARRQLKQMESQPLPGERARSTLQKEVHAAKSLAIIV GLFALCWLPLHIINCFTFFCPDCSHAPLWLMYLAIVLSHTNSVVNPFIY AYRIREFRQTFRKIIRSHVLRQQEPFKAAGTSARVLAAHGSDGEQVSL RLNGHPPGVWANGSAPHPERRPNGYALGLVSGGSAQESQGNTGLPD VELLSHELKGVCPEPPGLDDPLAQDGAGVS 26 Orexin MEPSATPGAQMGVPPGSREPSPVPPDYEDEFLRYLWRDYLYPKQYE OX1 WVLIAAYVAVFVVALVGNTLVCLAVWRNHHMRTVTNYFIVNLSLA DVLVTAICLPASLLVDITESWLFGHALCKVIPYLQAVSVSVAVLTLSFI ALDRWYAICHPLLFKSTARRARGSILGIWAVSLAIMVPQAAVMECSS VLPELANRTRLFSVCDERWADDLYPKIYHSCFFIVTYLAPLGLMAMA YFQIFRKLWGRQIPGTTSALVRNWKRPSDQLGDLEQGLSGEPQPRAR AFLAEVKQMRARRKTAKMLMVVLLVFALCYLPISVLNVLKRVFGMF RQASDREAVYACFTFSHWLVYANSAANPIIYNFLSGKFREQFKAAFSC CLPGLGPCGSLKAPSPRSSASHKSLSLQSRCSISKISEHVVLTSVTTVLP 27 Orexin MSGTKLEDSPPCRNWSSASELNETQEPFLNPTDYDDEEFLRYLWREY OX2 LHPKEYEWVLIAGYIIVFVVALIGNVLVCVAVWKNHHMRTVTNYFIV NLSLADVLVTITCLPATLVVDITETWFFGQSLCKVIPYLQTVSVSVSVL TLSCIALDRWYAICHPLMFKSTAKRARNSIVIIWIVSCIIMIPQAIVMEC STVFPGLANKTTLFTVCDERWGGEIYPKMYHICFFLVTYMAPLCLMV LAYLQIFRKLWCRQIPGTSSVVQRKWKPLQPVSQPRGPGQPTKSRMS AVAAEIKQIRARRKTARMLMIVLLVFAICYLPISILNVLKRVFGMFAH TEDRETVYAWFTFSHWLVYANSAANPIIYNFLSGKFREEFKAAFSCCC LGVHHRQEDRLTRGRTSTESRKSLTTQISNFDNISKLSEQVVLTSISTLP AANGAGPLQNW 28 PAR-2 IQGTNRSSKGRSLIGKVDGTSHVTGKGVTVETVFSVDEFSASVLTGKL TTVFLPIVYTIVFVVGLPSNGMALWVFLFRTKKKHPAVIYMANLALA DLLSVIWFPLKIAYHIHGNNWIYGEALCNVLIGFFYGNMYCSILFMTC LSVQRYWVIVNPMGHSRKKANIAIGISLAIWLLILLVTIPLYVVKQTIFI PALNITTCHDVLPEQLLVGDMFNYFLSLAIGVFLFPAFLTASAYVLMI RMLRSSAMDENSEKKRKRAIKLIVTVLAMYLICFTPSNLLLVVHYFLI KSQGQSHVYALYIVALCLSTLNSCIDPFVYYFVSHDFRDHAKNALLC RSVRTVKQMQVSLTSKKHSRKSSSYSSSSTTVKTSY 29 C3aR MASFSAETNSTDLLSQPWNEPPVILSMVILSLTFLLGLPGNGLVLWVA GLKMQRTVNTIWFLHLTLADLLCCLSLPFSLAHLALQGQWPYGRFLC KLIPSIIVLNMFASVFLLTAISLDRCLVVFKPIWCQNHRNVGMACSICG CIWVVAFVMCIPVFVYREIFTTDNHNRCGYKFGLSSSLDYPDFYGDPL ENRSLENIVQPPGEMNDRLDPSSFQTNDHPWTVPTVFQPQTFQRPSAD SLPRGSARLTSQNLYSNVFKPADVVSPKIPSGFPIEDHETSPLDNSDAF LSTHLKLFPSASSNSFYESELPQGFQDYYNLGQFTDDDQVPTPLVAITI TRLVVGFLLPSVIMIACYSFIVFRMQRGRFAKSQSKTFRVAVVVVAVF LVCWTPYHIFGVLSLLTDPETPLGKTLMSWDHVCIALASANSCFNPFL YALLGKDFRKKARQSIQGILEAAFSEELTRSTHCPSNNVISERNSTTV 30 LGR5 GSSPRSGVLLRGCPTHCHCEPDGRMLLRVDCSDLGLSELPSNLSVFTS YLDLSMNNISQLLPNPLPSLRFLEELRLAGNALTYIPKGAFTGLYSLKV LMLQNNQLRHVPTEALQNLRSLQSLRLDANHISYVPPSCFSGLHSLRH LWLDDNALTEIPVQAFRSLSALQAMTLALNKIHHIPDYAFGNLSSLVV LHLHNNRIHSLGKKCFDGLHSLETLDLNYNNLDEFPTAIRTLSNLKEL GFHSNNIRSIPEKAFVGNPSLITIHFYDNPIQFVGRSAFQHLPELRTLTL NGASQITEFPDLTGTANLESLTLTGAQISSLPQTVCNQLPNLQVLDLSY NLLEDLPSFSVCQKLQKIDLRHNEIYEIKVDTFQQLLSLRSLNLAWNKI AIIHPNAFSTLPSLIKLDLSSNLLSSFPITGLHGLTHLKLTGNHALQSLIS SENFPELKVIEMPYAYQCCAFGVCENAYKISNQWNKGDNSSMDDLH KKDAGMFQAQDERDLEDFLLDFEEDLKALHSVQCSPSPGPFKPCEHL LDGWLIRIGVWTIAVLALTCNALVTSTVFRSPLYISPIKLLIGVIAAVN MLTGVSSAVLAGVDAFTFGSFARHGAWWENGVGCHVIGFLSIFASES SVFLLTLAALERGFSVKYSAKFETKAPFSSLKVIILLCALLALTMAAVP LLGGSKYGASPLCLPLPFGEPSTMGYMVALILLNSLCFLMMTIAYTKL YCNLDKGDLENIWDCSMVKHIALLLFTNCILNCPVAFLSFSSLINLTFI SPEVIKFILLVVVPLPACLNPLLYILFNPHFKEDLVSLRKQTYVWTRSK HPSLMSINSDDVEKQSCDSTQALVTFTSSSITYDLPPSSVPSPAYPVTES CHLSSVAFVPCL 31 GPR101 MTSTCTNSTRESNSSHTCMPLSKMPISLAHGIIRSTVLVIFLAASFVGNI VLALVLQRKPQLLQVTNRFIFNLLVTDLLQISLVAPWVVATSVPLFWP LNSHFCTALVSLTHLFAFASVNTIVVVSVDRYLSIIHPLSYPSKMTQRR GYLLLYGTWIVAILQSTPPLYGWGQAAFDERNALCSMIWGASPSYTIL SVVSFIVIPLIVMIACYSVVFCAARRQHALLYNVKRHSLEVRVKDCVE NEDEEGAEKKEEFQDESEFRRQHEGEVKAKEGRMEAKDGSLKAKEG STGTSESSVEARGSEEVRESSTVASDGSMEGKEGSTKVEENSMKADK GRTEVNQCSIDLGEDDMEFGEDDINFSEDDVEAVNIPESLPPSRRNSNS NPPLPRCYQCKAAKVIFIIIFSYVLSLGPYCFLAVLAVWVDVETQVPQ WVITIIIWLFFLQCCIHPYVYGYMHKTIKKEIQDMLKKFFCKEKPPKED SHPDLPGTEGGTEGKIVPSYDSATFP 32 GPR151 MLAAAFADSNSSSMNVSFAHLHFAGGYLPSDSQDWRTIIPALLVAVC LVGFVGNLCVIGILLHNAWKGKPSMIHSLILNLSLADLSLLLFSAPIRA TAYSKSVWDLGWFVCKSSDWFIHTCMAAKSLTIVVVAKVCFMYASD PAKQVSIHNYTIWSVLVAIWTVASLLPLPEWFFSTIRHHEGVEMCLVD VPAVAEEFMSMFGKLYPLLAFGLPLFFASFYFWRAYDQCKKRGTKT QNLRNQIRSKQVTVMLLSIAIISALLWLPEWVAWLWVWHLKAAGPA PPQGFIALSQVLMFSISSANPLIFLVMSEEFREGLKGVWKWMITKKPPT VSESQETPAGNSEGLPDKVPSPESPASIPEKEKPSSPSSGKGKTEKAEIPI LPDVEQFWHERDTVPSVQDNDPIPWEHEDQETGEGVK 33 GPR161 MSLNSSLSCRKELSNLTEEEGGEGGVIITQFIAIIVITIFVCLGNLVIVVT LYKKSYLLTLSNKFVFSLTLSNFLLSVLVLPFVVTSSIRREWIFGVVWC NFSALLYLLISSASMLTLGVIAIDRYYAVLYPMVYPMKITGNRAVMA LVYIWLHSLIGCLPPLFGWSSVEFDEFKWMCVAAWHREPGYTAFWQI WCALFPFLVMLVCYGFIFRVARVKARKVHCGTVVIVEEDAQRTGRK NSSTSTSSSGSRRNAFQGVVYSANQCKALITILVVLGAFMVTWGPYM VVIASEALWGKSSVSPSLETWATWLSFASAVCHPLIYGLWNKTVRKE LLGMCFGDRYYREPFVQRQRTSRLFSISNRITDLGLSPHLTALMAGGQ PLGHSSSTGDTGFSCSQDSGTDMMLLEDYTSDDNPPSHCTCPPKRRSS VTFEDEVEQIKEAAKNSILHVKAEVHKSLDSYAASLAKAIEAEAKINL FGEEALPGVLVTARTVPGGGFGGRRGSRTLVSQRLQLQSIEEGDVLA AEQR 34 GPR17 MSKRSWWAGSRKPPREMLKLSGSDSSQSMNGLEVAPPGLITNFSLAT AEQCGQETPLENMLFASFYLLDFILALVGNTLALWLFIRDHKSGTPAN VFLMHLAVADLSCVLVLPTRLVYHFSGNHWPFGEIACRLTGFLFYLN MYASIYFLTCISADRFLAIVHPVKSLKLRRPLYAHLACAFLWVVVAV AMAPLLVSPQTVQTNHTVVCLQLYREKASHHALVSLAVAFTFPFITT VTCYLLIIRSLRQGLRVEKRLKTKAVRMIAIVLAIFLVCFVPYHVNRSV YVLHYRSHGASCATQRILALANRITSCLTSLNGALDPIMYFFVAEKFR HALCNLLCGKRLKGPPPSFEGKTNESSLSAKSEL 35 GPR183 MDIQMANNFTPPSATPQGNDCDLYAHHSTARIVMPLHYSLVFIIGLVG NLLALVVIVQNRKKINSTTLYSTNLVISDILFTTALPTRIAYYAMGFDW RIGDALCRITALVFYINTYAGVNFMTCLSIDRFIAVVHPLRYNKIKRIE HAKGVCIFVWILVFAQTLPLLINPMSKQEAERITCMEYPNFEETKSLP WILLGACFIGYVLPLIIILICYSQICCKLFRTAKQNPLTEKSGVNKKALN TIILIIVVFVLCFTPYHVAIIQHMIKKLRFSNFLECSQRHSFQISLHFTVC LMNFNCCMDPFIYFFACKGYKRKVMRMLKRQVSVSISSAVKSAPEEN SREMTETQMMIHSKSSNGK 36 CRTH2 MSANATLKPLCPILEQMSRLQSHSNTSIRYIDHAAVLLHGLASLLGLV ENGVILFVVGCRMRQTVVTTWVLHLALSDLLASASLPFFTYFLAVGH SWELGTTFCKLHSSIFFLNMFASGFLLSAISLDRCLQVVRPVWAQNHR TVAAAHKVCLVLWALAVLNTVPYFVFRDTISRLDGRIMCYYNVLLL NPGPDRDATCNSRQVALAVSKFLLAFLVPLAIIASSHAAVSLRLQHRG RRRPGRFVRLVAAVVAAFALCWGPYHVFSLLEARAHANPGLRPLVW RGLPFVTSLAFFNSVANPVLYVLTCPDMLRKLRRSLRTVLESVLVDDS ELGGAGSSRRRRTSSTARSASPLALCSRPEEPRGPARLLGWLLGSCAA SPQTGPLNRALSSTSS 37 5-HT4 MDKLDANVSSEEGFGSVEKVVLLTFLSTVILMAILGNLLVMVAVCW DRQLRKIKTNYFIVSLAFADLLVSVLVMPFGAIELVQDIWIYGEVFCL VRTSLDVLLTTASIFHLCCISLDRYYAICCQPLVYRNKMTPLRIALMLG GCWVIPTFISFLPIMQGWNNIGIIDLIEKRKFNQNSNSTYCVFMVNKPY AITCSVVAFYIPFLLMVLAYYRIYVTAKEHAHQIQMLQRAGASSESRP QSADQHSTHRMRTETKAAKTLCIIMGCFCLCWAPFFVTNIVDPFIDYT VPGQVWTAFLWLGYINSGLNPFLYAFLNKSFRRAFLIILCCDDERYRR PSILGQTVPCSTTTINGSTHVLRDAVECGGQWESQCHPPATSPLVAAQ PSDT 38 5-HT6 MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSL LIALICTQPALRNTSNFFLVSLFTSDLMVGLVVMPPAMLNALYGRWV LARGLCLLWTAFDVMCCSASILNLCLISLDRYLLILSPLRYKLRMTPL RALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPF VLVASGLTFFLPSGAICFTYCRILLAARKQAVQVASLTTGMASQASET LQVPRTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPF FVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRALG RFLPCPRCPRERQASLASPSLRTSHSGPRPGLSLQQVLPLPLPPDSDSDS DAGSGGSSGLRLTAQLLLPGEATQDPPLPTRAAAAVNFFNIDPAEPEL RPHPLGIPTN 39 CB2 MEECWVTEIANGSKDGLDSNPMKDYMILSGPQKTAVAVLCTLLGLL SALENVAVLYLILSSHQLRRKPSYLFIGSLAGADFLASVVFACSFVNFH VFHGVDSKAVFLLKIGSVTMTFTASVGSLLLTAIDRYLCLRYPPSYKA LLTRGRALVTLGIMWVLSALVSYLPLMGWTCCPRPCSELFPLIPNDYL LSWLLFIAFLFSGIIYTYGHVLWKAHQHVASLSGHQDRQVPGMARMR LDVRLAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVKKAFAFCS MLCLINSMVNPVIYALRSGEIRSSAHHCLAHWKKCVRGLGSEAKEEA PRSSVTETEADGKITPWPDSRDLDLSDC 40 Histamine-3 MERAPPDGPLNASGALAGEAAAAGGARGFSAAWTAVLAALMALLIV ATVLGNALVMLAFVADSSLRTQNNFFLLNLAISDFLVGAFCIPLYVPY VLTGRWTFGRGLCKLWLVVDYLLCTSSAFNIVLISYDRFLSVTRAVS YRAQQGDTRRAVRKMLLVWVLAFLLYGPAILSWEYLSGGSSIPEGHC YAEFFYNWYFLITASTLEFFTPFLSVTFFNLSIYLNIQRRTRLRLDGARE AAGPEPPPEAQPSPPPPPGCWGCWQKGHGEAMPLHRYGVGEAAVGA EAGEATLGGGGGGGSVASPTSSSGSSSRGTERPRSLKRGSKPSASSAS LEKRMKMVSQSFTQRFRLSRDRKVAKSLAVIVSIFGLCWAPYTLLMII RAACHGHCVPDYWYETSFWLLWANSAVNPVLYPLCHHSFRRAFTKL LCPQKLKIQPHSSLEHCWK 41 VPAC-1 or ARLQEECDYVQMIEVQHKQCLEEAQLENETIGCSKMWDNLTCWPAT VIPR1 PRGQVVVLACPLIFKLFSSIQGRNVSRSCTDEGWTHLEPGPYPIACGLD DKAASLDEQQTMFYGSVKTGYTIGYGLSLATLLVATAILSLFRKLHCT RNYIHMHLFISFILRAAAVFIKDLALFDSGESDQCSEGSVGCKAAMVF FQYCVMANFFWLLVEGLYLYTLLAVSFFSERKYFWGYILIGWGVPST FTMVWTIARIHFEDYGCWDTINSSLWWIIKGPILTSILVNFILFICIIRILL QKLRPPDIRKSDSSPYSRLARSTLLLIPLFGVHYIMFAFFPDNFKPEVK MVFELVVGSFQGFVVAILYCFLNGEVQAELRRKWRRWHLQGVLGW NPKYRHPSGGSNGATCSTQVSMLTRVSPGARRSSSFQAEVSLV 42 GIPR RAETGSKGQTAGELYQRWERYRRECQETLAAAEPPSGLACNGSFDM YVCWDYAAPNATARASCPWYLPWHHEIVAAGFVLRQCGSDGQWGL WRDHTQCENPEKNEAFLDQRLILERLQVMYTVGYSLSLATLLLALLIL SLFRRLHCTRNYIHINLFTSFMLRAAAILSRDRLLPRPGPYLGDQALAL WNQALAACRTAQIVTQYCVGANYTWLLVEGVYLHSLLVLVGGSEE GHFRYYLLLGWGAPALFVIPWVIVRYLYENTQCWERNEVKAIWWIIR TPILMTILINFLIFIRILGILLSKLRTRQMRCRDYRLRLARSTLTLVPLLG VHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQGFLVSVLYCFINKEV QSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSGSGPGEVPTSRGLS SGTLPGPGNEASRELESYC 43 5-HT1B MEEPGAQCAPPPPAGSETWVPQANLSSAPSQNCSAKDYIYQDSISLPW GPCR KVLLVMLLALITLATTLSNAFVIATVYRTRKLHTPANYLIASLAVTDL LVSILVMPISTMYTVTGRWTLGQVVCDFWLSSDITCCTASILHLCVIA LDRYWAITDAVEYSAKRTPKRAAVMIALVWVFSISISLPPFFWRQAK AEEEVSECVVNTDHILYTVYSTVGAFYFPTLLLIALYGRIYVEARSRIL KQTPNRTGKRLTRAQLITDSPGSTSSVTSINSRVPDVPSESGSPVYVNQ VKVRVSDALLEKKKLMAARERKATKTLGIILGAFIVCWLPFFIISLVM PICKDACWFHLAIFDFFTWLGYLNSLINPHYTMSNEDFKQAFHKLIRF KCTS 44 CCR7 QDEVTDDYIGDNTTVDYTLFESLCSKKDVRNFKAWFLPIMYSIICFVG LLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTLPFWAYSAA KSWVFGVHFCKLIFAIYKMSFFSGMLLLLCISIDRYVAIVQAVSAHRH RARVLLISKLSCVGIWILATVLSIPELLYSDLQRSSSEQAMRCSLITEHV EAFITIQVAQMVIGFLVPLLAMSFCYLVIIRTLLQARNFERNKAIKVIIA VVVVFIVFQLPYNGVVLAQTVANFNITSSTCELSKQLNIAYDVTYSLA CVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQEQLRQWSSCRHIRR SSMSVEAETTTTFSP 45 CXCR5 MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLM ASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAV ADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACI AVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVS QGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWC YVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLA RLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSD LSRLLTKLGCTGPASLCQLFPSWRRSSLSESENATSLTTF 46 GPR119 MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAV ADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRMAFVTSSAAASVLTVM LITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLGIPMF QQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIAS MHSQQIRKMEHAGAMAGGYRSPRTPSDFKALRTVSVLIGSFALSWTP FLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVR LQLYHMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG 47 GPR55 MSQQNTSGDCLFDGVNELMKTLQFAVHIPTFVLGLLLNLLAIHGFSTF LKNRWPDYAATSIYMINLAVFDLLLVLSLPFKMVLSQVQSPFPSLCTL VECLYFVSMYGSVFTICFISMDRFLAIRYPLLVSHLRSPRKIFGICCTIW VLVWTGSIPIYSFHGKVEKYMCFHNMSDDTWSAKVFFPLEVFGFLLP MGIMGFCCSRSIHILLGRRDHTQDWVQQKACIYSIAASLAVFVVSFLP VHLGFFLQFLVRNSFIVECRAKQSISFFLQLSMCFSNVNCCLDVFCYYF VIKEFRMNIRAHRPSRVQLVLQDTTISRG

Provided herein are scaffolds comprising GPCR binding domains, wherein the sequences of the GPCR binding domains support interaction with at least one GPCR. The sequence may be homologous or identical to a sequence of a GPCR ligand. In some instances, the GPCR binding domain sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some instances, the GPCR binding domain sequence comprises at least or about 95% homology to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some instances, the GPCR binding domain sequence comprises at least or about 97% homology to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some instances, the GPCR binding domain sequence comprises at least or about 99% homology to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some instances, the GPCR binding domain sequence comprises at least or about 100% homology to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47. In some instances, the GPCR binding domain sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47.

Libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains may bind to one or more GPCRs. In some instances, the scaffolds comprising GPCR binding domains binds to a single GPCR. In some instances, the scaffolds comprising GPCR binding domains binds to GPCRs in a same family or class. In some instances, the scaffolds comprising GPCR binding domains bind to multiple GPCRs. For example, the scaffolds are multimeric and comprise at least 2 scaffolds. In some instances, the multimeric scaffolds comprise at least or about 3, 4, 5, 6, 7, 8, or more than 8 scaffolds. In some instances, the multimeric scaffolds comprise at least 2 scaffolds linked by, for example, a dimerization domain, an amino acid linker, a disulfide bond, a chemical crosslink, or any other linker known in the art. In some instances, the multimeric scaffolds bind to the same GPCRs or different GPCRs.

Provided herein are GPCR binding libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains comprise variation in domain type, domain length, or residue variation. In some instances, the domain is a region in the scaffold comprising the GPCR binding domains. For example, the region is the VH, CDR-H3, or VL domain. In some instances, the domain is the GPCR binding domain.

Methods described herein provide for synthesis of a GPCR binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence. In some cases, the predetermined reference sequence is a nucleic acid sequence encoding for a protein, and the variant library comprises sequences encoding for variation of at least a single codon such that a plurality of different variants of a single residue in the subsequent protein encoded by the synthesized nucleic acid are generated by standard translation processes. In some instances, the GPCR binding library comprises varied nucleic acids collectively encoding variations at multiple positions. In some instances, the variant library comprises sequences encoding for variation of at least a single codon of a VH, CDR-H3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of at least a single codon in a GPCR binding domain. For example, at least one single codon of a GPCR binding domain as listed in Table 2 is varied. In some instances, the variant library comprises sequences encoding for variation of multiple codons of a VH, CDR-H3, or VL domain. In some instances, the variant library comprises sequences encoding for variation of multiple codons in a GPCR binding domain. An exemplary number of codons for variation include, but are not limited to, at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons.

Methods described herein provide for synthesis of a GPCR binding library of nucleic acids each encoding for a predetermined variant of at least one predetermined reference nucleic acid sequence, wherein the GPCR binding library comprises sequences encoding for variation of length of a domain. In some instances, the domain is VH, CDR-H3, or VL domain. In some instances, the domain is the GPCR binding domain. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 225, 250, 275, 300, or more than 300 codons less as compared to a predetermined reference sequence. In some instances, the library comprises sequences encoding for variation of length of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more than 300 codons more as compared to a predetermined reference sequence.

Provided herein are GPCR binding libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains, wherein the GPCR binding libraries are synthesized with various numbers of fragments. In some instances, the fragments comprise the VH, CDR-H3, or VL domain. In some instances, the GPCR binding libraries are synthesized with at least or about 2 fragments, 3 fragments, 4 fragments, 5 fragments, or more than 5 fragments. The length of each of the nucleic acid fragments or average length of the nucleic acids synthesized may be at least or about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or more than 600 base pairs. In some instances, the length is about 50 to 600, 75 to 575, 100 to 550, 125 to 525, 150 to 500, 175 to 475, 200 to 450, 225 to 425, 250 to 400, 275 to 375, or 300 to 350 base pairs.

GPCR binding libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains as described herein comprise various lengths of amino acids when translated. In some instances, the length of each of the amino acid fragments or average length of the amino acid synthesized may be at least or about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more than 150 amino acids. In some instances, the length of the amino acid is about 15 to 150, 20 to 145, 25 to 140, 30 to 135, 35 to 130, 40 to 125, 45 to 120, 50 to 115, 55 to 110, 60 to 110, 65 to 105, 70 to 100, or 75 to 95 amino acids. In some instances, the length of the amino acid is about 22 to about 75 amino acids.

GPCR binding libraries comprising de novo synthesized variant sequences encoding for scaffolds comprising GPCR binding domains comprise a number of variant sequences. In some instances, a number of variant sequences is de novo synthesized for a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3, VL, VH, or a combination thereof. In some instances, a number of variant sequences is de novo synthesized for framework element 1 (FW1), framework element 2 (FW2), framework element 3 (FW3), or framework element 4 (FW4). In some instances, a number of variant sequences is de novo synthesized for a GPCR binding domain. For example, the number of variant sequences is about 1 to about 10 sequences for the VH domain, about 10⁸ sequences for the GPCR binding domain, and about 1 to about 44 sequences for the VK domain. See FIG. 2. The number of variant sequences may be at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more than 500 sequences. In some instances, the number of variant sequences is about 10 to 300, 25 to 275, 50 to 250, 75 to 225, 100 to 200, or 125 to 150 sequences.

GPCR binding libraries comprising de novo synthesized variant sequences encoding for scaffolds comprising GPCR binding domains comprise improved diversity. For example, variants are generated by placing GPCR binding domain variants in immunoglobulin scaffold variants comprising N-terminal CDR-H3 variations and C-terminal CDR-H3 variations. In some instances, variants include affinity maturation variants. Alternatively or in combination, variants include variants in other regions of the immunoglobulin including, but not limited to, CDR-H1, CDR-H2, CDR-L1, CDR-L2, and CDR-L3. In some instances, the number of variants of the GPCR binding libraries is least or about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or more than 10¹⁰ non-identical sequences. For example, a library comprising about 10 variant sequences for a VH region, about 237 variant sequences for a CDR-H3 region, and about 43 variant sequences for a VL and CDR-L3 region comprises 10⁵ non-identical sequences (10×237×43). See FIGS. 4A-4B.

Following synthesis of GPCR binding libraries comprising nucleic acids encoding scaffolds comprising GPCR binding domains, libraries may be used for screening and analysis. For example, libraries are assayed for library displayability and panning. In some instances, displayability is assayed using a selectable tag. Exemplary tags include, but are not limited to, a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, an affinity tag or other labels or tags that are known in the art. In some instances, the tag is histidine, polyhistidine, myc, hemagglutinin (HA), or FLAG. For example as seen in FIG. 3, the GPCR binding libraries comprises nucleic acids encoding scaffolds comprising GPCR binding domains with multiple tags such as GFP, FLAG, and Lucy as well as a DNA barcode. In some instances, libraries are assayed by sequencing using various methods including, but not limited to, single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam-Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis.

Expression Systems

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains, wherein the libraries have improved specificity, stability, expression, folding, or downstream activity. In some instances, libraries described herein are used for screening and analysis.

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains, wherein the nucleic acid libraries are used for screening and analysis. In some instances, screening and analysis comprises in vitro, in vivo, or ex vivo assays. Cells for screening include primary cells taken from living subjects or cell lines. Cells may be from prokaryotes (e.g., bacteria and fungi) or eukaryotes (e.g., animals and plants). Exemplary animal cells include, without limitation, those from a mouse, rabbit, primate, and insect. In some instances, cells for screening include a cell line including, but not limited to, Chinese Hamster Ovary (CHO) cell line, human embryonic kidney (HEK) cell line, or baby hamster kidney (BHK) cell line. In some instances, nucleic acid libraries described herein may also be delivered to a multicellular organism. Exemplary multicellular organisms include, without limitation, a plant, a mouse, rabbit, primate, and insect.

Nucleic acid libraries described herein may be screened for various pharmacological or pharmacokinetic properties. In some instances, the libraries are screened using in vitro assays, in vivo assays, or ex vivo assays. For example, in vitro pharmacological or pharmacokinetic properties that are screened include, but are not limited to, binding affinity, binding specificity, and binding avidity. Exemplary in vivo pharmacological or pharmacokinetic properties of libraries described herein that are screened include, but are not limited to, therapeutic efficacy, activity, preclinical toxicity properties, clinical efficacy properties, clinical toxicity properties, immunogenicity, potency, and clinical safety properties.

Provided herein are nucleic acid libraries, wherein the nucleic acid libraries may be expressed in a vector. Expression vectors for inserting nucleic acid libraries disclosed herein may comprise eukaryotic or prokaryotic expression vectors. Exemplary expression vectors include, without limitation, mammalian expression vectors: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEFla-mCherry-N1 Vector, pEFla-tdTomato Vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-AN DNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1N5-His A and pDEST8. In some instances, the vector is pcDNA3 or pcDNA3.1.

Described herein are nucleic acid libraries that are expressed in a vector to generate a construct comprising a scaffold comprising sequences of GPCR binding domains. In some instances, a size of the construct varies. In some instances, the construct comprises at least or about 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some instances, a the construct comprises a range of about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 3,000 to 6,000, 3,000 to 7,000, 3,000 to 8,000, 3,000 to 9,000, 3,000 to 10,000, 4,000 to 5,000, 4,000 to 6,000, 4,000 to 7,000, 4,000 to 8,000, 4,000 to 9,000, 4,000 to 10,000, 5,000 to 6,000, 5,000 to 7,000, 5,000 to 8,000, 5,000 to 9,000, 5,000 to 10,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 9,000, 6,000 to 10,000, 7,000 to 8,000, 7,000 to 9,000, 7,000 to 10,000, 8,000 to 9,000, 8,000 to 10,000, or 9,000 to 10,000 bases.

Provided herein are libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains, wherein the nucleic acid libraries are expressed in a cell. In some instances, the libraries are synthesized to express a reporter gene. Exemplary reporter genes include, but are not limited to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), cerulean fluorescent protein, citrine fluorescent protein, orange fluorescent protein, cherry fluorescent protein, turquoise fluorescent protein, blue fluorescent protein, horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, and derivatives thereof. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g. fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.

Diseases and Disorders

Provided herein are GPCR binding libraries comprising nucleic acids encoding for scaffolds comprising GPCR binding domains may have therapeutic effects. In some instances, the GPCR binding libraries result in protein when translated that is used to treat a disease or disorder. In some instances, the protein is an immunoglobulin. In some instances, the protein is a peptidomimetic. Exemplary diseases include, but are not limited to, cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, or a neurological disease or disorder. In some instances, the cancer is a solid cancer or a hematologic cancer. In some instances, an inhibitor of GPCR glucagon like peptide 1 receptor (GLP1R) as described herein is used for treatment of a metabolic disorder. In some instances, an inhibitor of GPCR GLP1R as described herein is used for treatment of weight gain (or for inducing weight loss), treatment of obesity, or treatment of Type II diabetes. In some instances, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously. In some instances, a pharmaceutical composition comprises an antibody or antibody fragment described herein comprising a CDR-H3 comprising a sequence of any one of SEQ ID NOS: 2420 to 2436. In further instances, the pharmaceutical composition is used for treatment of a metabolic disorder.

Variant Libraries

Codon Variation

Variant nucleic acid libraries described herein may comprise a plurality of nucleic acids, wherein each nucleic acid encodes for a variant codon sequence compared to a reference nucleic acid sequence. In some instances, each nucleic acid of a first nucleic acid population contains a variant at a single variant site. In some instances, the first nucleic acid population contains a plurality of variants at a single variant site such that the first nucleic acid population contains more than one variant at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding multiple codon variants at the same variant site. The first nucleic acid population may comprise nucleic acids collectively encoding up to 19 or more codons at the same position. The first nucleic acid population may comprise nucleic acids collectively encoding up to 60 variant triplets at the same position, or the first nucleic acid population may comprise nucleic acids collectively encoding up to 61 different triplets of codons at the same position. Each variant may encode for a codon that results in a different amino acid during translation. Table 3 provides a listing of each codon possible (and the representative amino acid) for a variant site.

TABLE 3 List of codons and amino acids One Three letter letter Amino Acids code code Codons Alanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E Glu GAA GAG Phenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGT Histidine H His CAC CAT Isoleucine I Iso ATA ATC ATT Lysine K Lys AAA AAG Leucine L Leu TTA TTG CTA CTC CTG CTT Methionine M Met ATG Asparagine N Asn AAC AAT Proline P Pro CCA CCC CCG CCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y Tyr TAC TAT

A nucleic acid population may comprise varied nucleic acids collectively encoding up to 20 codon variations at multiple positions. In such cases, each nucleic acid in the population comprises variation for codons at more than one position in the same nucleic acid. In some instances, each nucleic acid in the population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more codons in a single nucleic acid. In some instances, each variant long nucleic acid comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single long nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more codons in a single nucleic acid. In some instances, the variant nucleic acid population comprises variation for codons in at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more codons in a single long nucleic acid.

Highly Parallel Nucleic Acid Synthesis

Provided herein is a platform approach utilizing miniaturization, parallelization, and vertical integration of the end-to-end process from polynucleotide synthesis to gene assembly within nanowells on silicon to create a revolutionary synthesis platform. Devices described herein provide, with the same footprint as a 96-well plate, a silicon synthesis platform is capable of increasing throughput by a factor of up to 1,000 or more compared to traditional synthesis methods, with production of up to approximately 1,000,000 or more polynucleotides, or 10,000 or more genes in a single highly-parallelized run.

With the advent of next-generation sequencing, high resolution genomic data has become an important factor for studies that delve into the biological roles of various genes in both normal biology and disease pathogenesis. At the core of this research is the central dogma of molecular biology and the concept of “residue-by-residue transfer of sequential information.” Genomic information encoded in the DNA is transcribed into a message that is then translated into the protein that is the active product within a given biological pathway.

Another exciting area of study is on the discovery, development and manufacturing of therapeutic molecules focused on a highly-specific cellular target. High diversity DNA sequence libraries are at the core of development pipelines for targeted therapeutics. Gene mutants are used to express proteins in a design, build, and test protein engineering cycle that ideally culminates in an optimized gene for high expression of a protein with high affinity for its therapeutic target. As an example, consider the binding pocket of a receptor. The ability to test all sequence permutations of all residues within the binding pocket simultaneously will allow for a thorough exploration, increasing chances of success. Saturation mutagenesis, in which a researcher attempts to generate all possible mutations at a specific site within the receptor, represents one approach to this development challenge. Though costly and time and labor-intensive, it enables each variant to be introduced into each position. In contrast, combinatorial mutagenesis, where a few selected positions or short stretch of DNA may be modified extensively, generates an incomplete repertoire of variants with biased representation.

To accelerate the drug development pipeline, a library with the desired variants available at the intended frequency in the right position available for testing—in other words, a precision library, enables reduced costs as well as turnaround time for screening. Provided herein are methods for synthesizing nucleic acid synthetic variant libraries which provide for precise introduction of each intended variant at the desired frequency. To the end user, this translates to the ability to not only thoroughly sample sequence space but also be able to query these hypotheses in an efficient manner, reducing cost and screening time. Genome-wide editing can elucidate important pathways, libraries where each variant and sequence permutation can be tested for optimal functionality, and thousands of genes can be used to reconstruct entire pathways and genomes to re-engineer biological systems for drug discovery.

In a first example, a drug itself can be optimized using methods described herein. For example, to improve a specified function of an antibody, a variant polynucleotide library encoding for a portion of the antibody is designed and synthesized. A variant nucleic acid library for the antibody can then be generated by processes described herein (e.g., PCR mutagenesis followed by insertion into a vector). The antibody is then expressed in a production cell line and screened for enhanced activity. Example screens include examining modulation in binding affinity to an antigen, stability, or effector function (e.g., ADCC, complement, or apoptosis). Exemplary regions to optimize the antibody include, without limitation, the Fc region, Fab region, variable region of the Fab region, constant region of the Fab region, variable domain of the heavy chain or light chain (V_(H) or V_(L)), and specific complementarity-determining regions (CDRs) of V_(H) or V_(L).

Nucleic acid libraries synthesized by methods described herein may be expressed in various cells associated with a disease state. Cells associated with a disease state include cell lines, tissue samples, primary cells from a subject, cultured cells expanded from a subject, or cells in a model system. Exemplary model systems include, without limitation, plant and animal models of a disease state.

To identify a variant molecule associated with prevention, reduction or treatment of a disease state, a variant nucleic acid library described herein is expressed in a cell associated with a disease state, or one in which a cell a disease state can be induced. In some instances, an agent is used to induce a disease state in cells. Exemplary tools for disease state induction include, without limitation, a Cre/Lox recombination system, LPS inflammation induction, and streptozotocin to induce hypoglycemia. The cells associated with a disease state may be cells from a model system or cultured cells, as well as cells from a subject having a particular disease condition. Exemplary disease conditions include a bacterial, fungal, viral, autoimmune, or proliferative disorder (e.g., cancer). In some instances, the variant nucleic acid library is expressed in the model system, cell line, or primary cells derived from a subject, and screened for changes in at least one cellular activity. Exemplary cellular activities include, without limitation, proliferation, cycle progression, cell death, adhesion, migration, reproduction, cell signaling, energy production, oxygen utilization, metabolic activity, and aging, response to free radical damage, or any combination thereof.

Substrates

Devices used as a surface for polynucleotide synthesis may be in the form of substrates which include, without limitation, homogenous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support the attachment and synthesis of polynucleotides. In some instances, substrates comprise a homogenous array surface. For example, the homogenous array surface is a homogenous plate. The term “locus” as used herein refers to a discrete region on a structure which provides support for polynucleotides encoding for a single predetermined sequence to extend from the surface. In some instances, a locus is on a two dimensional surface, e.g., a substantially planar surface. In some instances, a locus is on a three-dimensional surface, e.g., a well, microwell, channel, or post. In some instances, a surface of a locus comprises a material that is actively functionalized to attach to at least one nucleotide for polynucleotide synthesis, or preferably, a population of identical nucleotides for synthesis of a population of polynucleotides. In some instances, polynucleotide refers to a population of polynucleotides encoding for the same nucleic acid sequence. In some cases, a surface of a substrate is inclusive of one or a plurality of surfaces of a substrate. The average error rates for polynucleotides synthesized within a library described here using the systems and methods provided are often less than 1 in 1000, less than about 1 in 2000, less than about 1 in 3000 or less often without error correction.

Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some instances, a substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the surfaces provide support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the substrate provides a surface environment for the growth of polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more.

Provided herein are methods for polynucleotide synthesis on distinct loci of a substrate, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, each polynucleotide sequence is synthesized with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more redundancy across different loci within the same cluster of loci on a surface for polynucleotide synthesis. In some instances, the loci of a substrate are located within a plurality of clusters. In some instances, a substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a substrate comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some instances, a substrate comprises about 10,000 distinct loci. The amount of loci within a single cluster is varied in different instances. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more loci. In some instances, each cluster includes about 50-500 loci. In some instances, each cluster includes about 100-200 loci. In some instances, each cluster includes about 100-150 loci. In some instances, each cluster includes about 109, 121, 130 or 137 loci. In some instances, each cluster includes about 19, 20, 61, 64 or more loci. Alternatively or in combination, polynucleotide synthesis occurs on a homogenous array surface.

In some instances, the number of distinct polynucleotides synthesized on a substrate is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster or surface of a substrate is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1,000 or more loci per mm². In some cases, a substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200 mm². In some instances, the distance between the centers of two adjacent loci within a cluster or surface is from about 10-500, from about 10-200, or from about 10-100 um. In some instances, the distance between two centers of adjacent loci is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some instances, the distance between the centers of two adjacent loci is less than about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, each locus has a width of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 um. In some cases, each locus is has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some instances, the density of clusters within a substrate is at least or about 1 cluster per 100 mm², 1 cluster per 10 mm², 1 cluster per 5 mm², 1 cluster per 4 mm², 1 cluster per 3 mm², 1 cluster per 2 mm², 1 cluster per 1 mm², 2 clusters per 1 mm², 3 clusters per 1 mm², 4 clusters per 1 mm², 5 clusters per 1 mm², 10 clusters per 1 mm², 50 clusters per 1 mm² or more. In some instances, a substrate comprises from about 1 cluster per 10 mm² to about 10 clusters per 1 mm². In some instances, the distance between the centers of two adjacent clusters is at least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is between about 50-100, 50-200, 50-300, 50-500, and 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster has a cross section of about 0.5 to about 2, about 0.5 to about 1, or about 1 to about 2 mm. In some cases, each cluster has a cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm. In some cases, each cluster has an interior cross section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mm.

In some instances, a substrate is about the size of a standard 96 well plate, for example between about 100 and about 200 mm by between about 50 and about 150 mm. In some instances, a substrate has a diameter less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100 or 50 mm. In some instances, the diameter of a substrate is between about 25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In some instances, a substrate has a planar surface area of at least about 100; 200; 500; 1,000; 2,000; 5,000; 10,000; 12,000; 15,000; 20,000; 30,000; 40,000; 50,000 mm² or more. In some instances, the thickness of a substrate is between about 50-2000, 50-1000, 100-1000, 200-1000, or 250-1000 mm.

Surface Materials

Substrates, devices, and reactors provided herein are fabricated from any variety of materials suitable for the methods, compositions, and systems described herein. In certain instances, substrate materials are fabricated to exhibit a low level of nucleotide binding. In some instances, substrate materials are modified to generate distinct surfaces that exhibit a high level of nucleotide binding. In some instances, substrate materials are transparent to visible and/or UV light. In some instances, substrate materials are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of a substrate. In some instances, conductive materials are connected to an electric ground. In some instances, the substrate is heat conductive or insulated. In some instances, the materials are chemical resistant and heat resistant to support chemical or biochemical reactions, for example polynucleotide synthesis reaction processes. In some instances, a substrate comprises flexible materials. For flexible materials, materials can include, without limitation: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like. In some instances, a substrate comprises rigid materials. For rigid materials, materials can include, without limitation: glass; fuse silica; silicon, plastics (for example polytetraflouroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The substrate, solid support or reactors can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.

Surface Architecture

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some instances, a substrate comprises raised and/or lowered features. One benefit of having such features is an increase in surface area to support polynucleotide synthesis. In some instances, a substrate having raised and/or lowered features is referred to as a three-dimensional substrate. In some cases, a three-dimensional substrate comprises one or more channels. In some cases, one or more loci comprise a channel. In some cases, the channels are accessible to reagent deposition via a deposition device such as a material deposition device. In some cases, reagents and/or fluids collect in a larger well in fluid communication one or more channels. For example, a substrate comprises a plurality of channels corresponding to a plurality of loci with a cluster, and the plurality of channels are in fluid communication with one well of the cluster. In some methods, a library of polynucleotides is synthesized in a plurality of loci of a cluster.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some instances, the structure is configured to allow for controlled flow and mass transfer paths for polynucleotide synthesis on a surface. In some instances, the configuration of a substrate allows for the controlled and even distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some instances, the configuration of a substrate allows for increased sweep efficiency, for example by providing sufficient volume for a growing polynucleotide such that the excluded volume by the growing polynucleotide does not take up more than 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%, or less of the initially available volume that is available or suitable for growing the polynucleotide. In some instances, a three-dimensional structure allows for managed flow of fluid to allow for the rapid exchange of chemical exposure.

Provided herein are substrates for the methods, compositions, and systems described herein, wherein the substrates comprise structures suitable for the methods, compositions, and systems described herein. In some instances, segregation is achieved by physical structure. In some instances, segregation is achieved by differential functionalization of the surface generating active and passive regions for polynucleotide synthesis. In some instances, differential functionalization is achieved by alternating the hydrophobicity across the substrate surface, thereby creating water contact angle effects that cause beading or wetting of the deposited reagents. Employing larger structures can decrease splashing and cross-contamination of distinct polynucleotide synthesis locations with reagents of the neighboring spots. In some cases, a device, such as a material deposition device, is used to deposit reagents to distinct polynucleotide synthesis locations. Substrates having three-dimensional features are configured in a manner that allows for the synthesis of a large number of polynucleotides (e.g., more than about 10,000) with a low error rate (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000, 1:3,000, 1:5,000, or 1:10,000). In some cases, a substrate comprises features with a density of about or greater than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400 or 500 features per mm².

A well of a substrate may have the same or different width, height, and/or volume as another well of the substrate. A channel of a substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some instances, the diameter of a cluster or the diameter of a well comprising a cluster, or both, is between about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-10, 0.5-5, or 0.5-2 mm. In some instances, the diameter of a cluster or well or both is less than or about 5, 4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some instances, the diameter of a cluster or well or both is between about 1.0 and 1.3 mm. In some instances, the diameter of a cluster or well, or both is about 1.150 mm. In some instances, the diameter of a cluster or well, or both is about 0.08 mm. The diameter of a cluster refers to clusters within a two-dimensional or three-dimensional substrate.

In some instances, the height of a well is from about 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, or 500-1000 um. In some cases, the height of a well is less than about 1000, 900, 800, 700, or 600 um.

In some instances, a substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of a channel is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, or 10-50 um. In some cases, the height of a channel is less than 100, 80, 60, 40, or 20 um.

In some instances, the diameter of a channel, locus (e.g., in a substantially planar substrate) or both channel and locus (e.g., in a three-dimensional substrate wherein a locus corresponds to a channel) is from about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100 um, for example, about 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the diameter of a channel, locus, or both channel and locus is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 um. In some instances, the distance between the center of two adjacent channels, loci, or channels and loci is from about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example, about 20 um.

Surface Modifications

Provided herein are methods for polynucleotide synthesis on a surface, wherein the surface comprises various surface modifications. In some instances, the surface modifications are employed for the chemical and/or physical alteration of a surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of a substrate surface. For example, surface modifications include, without limitation, (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e., providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e., removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface.

In some cases, the addition of a chemical layer on top of a surface (referred to as adhesion promoter) facilitates structured patterning of loci on a surface of a substrate. Exemplary surfaces for application of adhesion promotion include, without limitation, glass, silicon, silicon dioxide and silicon nitride. In some cases, the adhesion promoter is a chemical with a high surface energy. In some instances, a second chemical layer is deposited on a surface of a substrate. In some cases, the second chemical layer has a low surface energy. In some cases, surface energy of a chemical layer coated on a surface supports localization of droplets on the surface. Depending on the patterning arrangement selected, the proximity of loci and/or area of fluid contact at the loci are alterable.

In some instances, a substrate surface, or resolved loci, onto which nucleic acids or other moieties are deposited, e.g., for polynucleotide synthesis, are smooth or substantially planar (e.g., two-dimensional) or have irregularities, such as raised or lowered features (e.g., three-dimensional features). In some instances, a substrate surface is modified with one or more different layers of compounds. Such modification layers of interest include, without limitation, inorganic and organic layers such as metals, metal oxides, polymers, small organic molecules and the like.

In some instances, resolved loci of a substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, a moiety is chemically inert. In some cases, a moiety is configured to support a desired chemical reaction, for example, one or more processes in a polynucleotide synthesis reaction. The surface energy, or hydrophobicity, of a surface is a factor for determining the affinity of a nucleotide to attach onto the surface. In some instances, a method for substrate functionalization comprises: (a) providing a substrate having a surface that comprises silicon dioxide; and (b) silanizing the surface using, a suitable silanizing agent described herein or otherwise known in the art, for example, an organofunctional alkoxysilane molecule. Methods and functionalizing agents are described in U.S. Pat. No. 5,474,796, which is herein incorporated by reference in its entirety.

In some instances, a substrate surface is functionalized by contact with a derivatizing composition that contains a mixture of silanes, under reaction conditions effective to couple the silanes to the substrate surface, typically via reactive hydrophilic moieties present on the substrate surface. Silanization generally covers a surface through self-assembly with organofunctional alkoxysilane molecules. A variety of siloxane functionalizing reagents can further be used as currently known in the art, e.g., for lowering or increasing surface energy. The organofunctional alkoxysilanes are classified according to their organic functions.

Polynucleotide Synthesis

Methods of the current disclosure for polynucleotide synthesis may include processes involving phosphoramidite chemistry. In some instances, polynucleotide synthesis comprises coupling a base with phosphoramidite. Polynucleotide synthesis may comprise coupling a base by deposition of phosphoramidite under coupling conditions, wherein the same base is optionally deposited with phosphoramidite more than once, i.e., double coupling. Polynucleotide synthesis may comprise capping of unreacted sites. In some instances, capping is optional. Polynucleotide synthesis may also comprise oxidation or an oxidation step or oxidation steps. Polynucleotide synthesis may comprise deblocking, detritylation, and sulfurization. In some instances, polynucleotide synthesis comprises either oxidation or sulfurization. In some instances, between one or each step during a polynucleotide synthesis reaction, the device is washed, for example, using tetrazole or acetonitrile. Time frames for any one step in a phosphoramidite synthesis method may be less than about 2 min, 1 min, 50 sec, 40 sec, 30 sec, 20 sec and 10 sec.

Polynucleotide synthesis using a phosphoramidite method may comprise a subsequent addition of a phosphoramidite building block (e.g., nucleoside phosphoramidite) to a growing polynucleotide chain for the formation of a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3′ to 5′ direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid chain per synthesis cycle. In some instances, each synthesis cycle comprises a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester linkage between an activated nucleoside phosphoramidite and a nucleoside bound to the substrate, for example, via a linker. In some instances, the nucleoside phosphoramidite is provided to the device activated. In some instances, the nucleoside phosphoramidite is provided to the device with an activator. In some instances, nucleoside phosphoramidites are provided to the device in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition of a nucleoside phosphoramidite, the device is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the device is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. A common protecting group is 4,4′-dimethoxytrityl (DMT).

Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step is useful to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole may react, to a small extent, with the 06 position of guanosine. Without being bound by theory, upon oxidation with I₂/water, this side product, possibly via 06-N7 migration, may undergo depurination. The apurinic sites may end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The O6 modifications may be removed by treatment with the capping reagent prior to oxidation with I₂/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the device is optionally washed.

In some instances, following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, the device bound growing nucleic acid is oxidized. The oxidation step comprises the phosphite triester is oxidized into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). Oxidation may be carried out under anhydrous conditions using, e.g. tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for device drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the device and growing polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including but not limited to 3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

In order for a subsequent cycle of nucleoside incorporation to occur through coupling, the protected 5′ end of the device bound growing polynucleotide is removed so that the primary hydroxyl group is reactive with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions of the disclosure described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the device bound polynucleotide is washed after deblocking. In some instances, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.

Methods for the synthesis of polynucleotides typically involve an iterating sequence of the following steps: application of a protected monomer to an actively functionalized surface (e.g., locus) to link with either the activated surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it is reactive with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.

Methods for phosphoramidite-based polynucleotide synthesis comprise a series of chemical steps. In some instances, one or more steps of a synthesis method involve reagent cycling, where one or more steps of the method comprise application to the device of a reagent useful for the step. For example, reagents are cycled by a series of liquid deposition and vacuum drying steps. For substrates comprising three-dimensional features such as wells, microwells, channels and the like, reagents are optionally passed through one or more regions of the device via the wells and/or channels.

Methods and systems described herein relate to polynucleotide synthesis devices for the synthesis of polynucleotides. The synthesis may be in parallel. For example, at least or about at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 10000, 50000, 75000, 100000 or more polynucleotides can be synthesized in parallel. The total number polynucleotides that may be synthesized in parallel may be from 2-100000, 3-50000, 4-10000, 5-1000, 6-900, 7-850, 8-800, 9-750, 10-700, 11-650, 12-600, 13-550, 14-500, 15-450, 16-400, 17-350, 18-300, 19-250, 20-200, 21-150, 22-100, 23-50, 24-45, 25-40, 30-35. Those of skill in the art appreciate that the total number of polynucleotides synthesized in parallel may fall within any range bound by any of these values, for example 25-100. The total number of polynucleotides synthesized in parallel may fall within any range defined by any of the values serving as endpoints of the range. Total molar mass of polynucleotides synthesized within the device or the molar mass of each of the polynucleotides may be at least or at least about 10, 20, 30, 40, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000 picomoles, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at least or about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500 nucleotides, or more. The length of each of the polynucleotides or average length of the polynucleotides within the device may be at most or about at most 500, 400, 300, 200, 150, 100, 50, 45, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 nucleotides, or less. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall from 10-500, 9-400, 11-300, 12-200, 13-150, 14-100, 15-50, 16-45, 17-40, 18-35, 19-25. Those of skill in the art appreciate that the length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range bound by any of these values, for example 100-300. The length of each of the polynucleotides or average length of the polynucleotides within the device may fall within any range defined by any of the values serving as endpoints of the range.

Methods for polynucleotide synthesis on a surface provided herein allow for synthesis at a fast rate. As an example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 nucleotides per hour, or more are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified versions thereof. In some instances, libraries of polynucleotides are synthesized in parallel on substrate. For example, a device comprising about or at least about 100; 1,000; 10,000; 30,000; 75,000; 100,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or 5,000,000 resolved loci is able to support the synthesis of at least the same number of distinct polynucleotides, wherein polynucleotide encoding a distinct sequence is synthesized on a resolved locus. In some instances, a library of polynucleotides is synthesized on a device with low error rates described herein in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less. In some instances, larger nucleic acids assembled from a polynucleotide library synthesized with low error rate using the substrates and methods described herein are prepared in less than about three months, two months, one month, three weeks, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days, 24 hours or less.

In some instances, methods described herein provide for generation of a library of nucleic acids comprising variant nucleic acids differing at a plurality of codon sites. In some instances, a nucleic acid may have 1 site, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites 18 sites, 19 sites, 20 sites, 30 sites, 40 sites, 50 sites, or more of variant codon sites.

In some instances, the one or more sites of variant codon sites may be adjacent. In some instances, the one or more sites of variant codon sites may not be adjacent and separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more codons.

In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein all the variant codon sites are adjacent to one another, forming a stretch of variant codon sites. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein none the variant codon sites are adjacent to one another. In some instances, a nucleic acid may comprise multiple sites of variant codon sites, wherein some the variant codon sites are adjacent to one another, forming a stretch of variant codon sites, and some of the variant codon sites are not adjacent to one another.

Referring to the Figures, FIG. 7 illustrates an exemplary process workflow for synthesis of nucleic acids (e.g., genes) from shorter nucleic acids. The workflow is divided generally into phases: (1) de novo synthesis of a single stranded nucleic acid library, (2) joining nucleic acids to form larger fragments, (3) error correction, (4) quality control, and (5) shipment. Prior to de novo synthesis, an intended nucleic acid sequence or group of nucleic acid sequences is preselected. For example, a group of genes is preselected for generation.

Once large nucleic acids for generation are selected, a predetermined library of nucleic acids is designed for de novo synthesis. Various suitable methods are known for generating high density polynucleotide arrays. In the workflow example, a device surface layer is provided. In the example, chemistry of the surface is altered in order to improve the polynucleotide synthesis process. Areas of low surface energy are generated to repel liquid while areas of high surface energy are generated to attract liquids. The surface itself may be in the form of a planar surface or contain variations in shape, such as protrusions or microwells which increase surface area. In the workflow example, high surface energy molecules selected serve a dual function of supporting DNA chemistry, as disclosed in International Patent Application Publication WO/2015/021080, which is herein incorporated by reference in its entirety.

In situ preparation of polynucleotide arrays is generated on a solid support and utilizes single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 702. In some instances, polynucleotides are cleaved from the surface at this stage. Cleavage includes gas cleavage, e.g., with ammonia or methylamine.

The generated polynucleotide libraries are placed in a reaction chamber. In this exemplary workflow, the reaction chamber (also referred to as “nanoreactor”) is a silicon coated well, containing PCR reagents and lowered onto the polynucleotide library 703. Prior to or after the sealing 704 of the polynucleotides, a reagent is added to release the polynucleotides from the substrate. In the exemplary workflow, the polynucleotides are released subsequent to sealing of the nanoreactor 705. Once released, fragments of single stranded polynucleotides hybridize in order to span an entire long range sequence of DNA. Partial hybridization 705 is possible because each synthesized polynucleotide is designed to have a small portion overlapping with at least one other polynucleotide in the pool.

After hybridization, a PCA reaction is commenced. During the polymerase cycles, the polynucleotides anneal to complementary fragments and gaps are filled in by a polymerase. Each cycle increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double stranded DNA 706.

After PCA is complete, the nanoreactor is separated from the device 707 and positioned for interaction with a device having primers for PCR 708. After sealing, the nanoreactor is subject to PCR 709 and the larger nucleic acids are amplified. After PCR 710, the nanochamber is opened 711, error correction reagents are added 712, the chamber is sealed 713 and an error correction reaction occurs to remove mismatched base pairs and/or strands with poor complementarity from the double stranded PCR amplification products 714. The nanoreactor is opened and separated 715. Error corrected product is next subject to additional processing steps, such as PCR and molecular bar coding, and then packaged 722 for shipment 723.

In some instances, quality control measures are taken. After error correction, quality control steps include for example interaction with a wafer having sequencing primers for amplification of the error corrected product 716, sealing the wafer to a chamber containing error corrected amplification product 717, and performing an additional round of amplification 718. The nanoreactor is opened 719 and the products are pooled 720 and sequenced 721. After an acceptable quality control determination is made, the packaged product 722 is approved for shipment 723.

In some instances, a nucleic acid generate by a workflow such as that in FIG. 7 is subject to mutagenesis using overlapping primers disclosed herein. In some instances, a library of primers are generated by in situ preparation on a solid support and utilize single nucleotide extension process to extend multiple oligomers in parallel. A deposition device, such as a material deposition device, is designed to release reagents in a step wise fashion such that multiple polynucleotides extend, in parallel, one residue at a time to generate oligomers with a predetermined nucleic acid sequence 702.

Computer Systems

Any of the systems described herein, may be operably linked to a computer and may be automated through a computer either locally or remotely. In various instances, the methods and systems of the disclosure may further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the disclosure. The computer systems may be programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.

The computer system 800 illustrated in FIG. 8 may be understood as a logical apparatus that can read instructions from media 811 and/or a network port 805, which can optionally be connected to server 809 having fixed media 812. The system, such as shown in FIG. 8 can include a CPU 801, disk drives 803, optional input devices such as keyboard 815 and/or mouse 816 and optional monitor 807. Data communication can be achieved through the indicated communication medium to a server at a local or a remote location. The communication medium can include any means of transmitting and/or receiving data. For example, the communication medium can be a network connection, a wireless connection or an internet connection. Such a connection can provide for communication over the World Wide Web. It is envisioned that data relating to the present disclosure can be transmitted over such networks or connections for reception and/or review by a party 822 as illustrated in FIG. 8.

FIG. 14 is a block diagram illustrating a first example architecture of a computer system 1400 that can be used in connection with example instances of the present disclosure. As depicted in FIG. 14, the example computer system can include a processor 1402 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 Apple A4™ processor, Marvell PXA 930™ processor, or a functionally-equivalent processor. Multiple threads of execution can be used for parallel processing. In some instances, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across systems over a network comprising a plurality of computers, cell phones, and/or personal data assistant devices.

As illustrated in FIG. 9, a high speed cache 904 can be connected to, or incorporated in, the processor 902 to provide a high speed memory for instructions or data that have been recently, or are frequently, used by processor 902. The processor 902 is connected to a north bridge 906 by a processor bus 908. The north bridge 906 is connected to random access memory (RAM) 910 by a memory bus 912 and manages access to the RAM 910 by the processor 902. The north bridge 906 is also connected to a south bridge 914 by a chipset bus 916. The south bridge 914 is, in turn, connected to a peripheral bus 918. The peripheral bus can be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 918. In some alternative architectures, the functionality of the north bridge can be incorporated into the processor instead of using a separate north bridge chip. In some instances, system 900 can include an accelerator card 922 attached to the peripheral bus 918. The accelerator can include field programmable gate arrays (FPGAs) or other hardware for accelerating certain processing. For example, an accelerator can be used for adaptive data restructuring or to evaluate algebraic expressions used in extended set processing.

Software and data are stored in external storage 924 and can be loaded into RAM 910 and/or cache 904 for use by the processor. The system 900 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example instances of the present disclosure. In this example, system 900 also includes network interface cards (NICs) 920 and 921 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.

FIG. 10 is a diagram showing a network 1000 with a plurality of computer systems 1002 a, and 1002 b, a plurality of cell phones and personal data assistants 1002 c, and Network Attached Storage (NAS) 1004 a, and 1004 b. In example instances, systems 1002 a, 1002 b, and 1002 c can manage data storage and optimize data access for data stored in Network Attached Storage (NAS) 1004 a and 1004 b. A mathematical model can be used for the data and be evaluated using distributed parallel processing across computer systems 1002 a, and 1002 b, and cell phone and personal data assistant systems 1002 c. Computer systems 1002 a, and 1002 b, and cell phone and personal data assistant systems 1002 c can also provide parallel processing for adaptive data restructuring of the data stored in Network Attached Storage (NAS) 1004 a and 1004 b. FIG. 10 illustrates an example only, and a wide variety of other computer architectures and systems can be used in conjunction with the various instances of the present disclosure. For example, a blade server can be used to provide parallel processing. Processor blades can be connected through a back plane to provide parallel processing. Storage can also be connected to the back plane or as Network Attached Storage (NAS) through a separate network interface. In some example instances, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other instances, some or all of the processors can use a shared virtual address memory space.

FIG. 11 is a block diagram of a multiprocessor computer system 1100 using a shared virtual address memory space in accordance with an example instance. The system includes a plurality of processors 1102 a-f that can access a shared memory subsystem 1104. The system incorporates a plurality of programmable hardware memory algorithm processors (MAPs) 1106 a-f in the memory subsystem 1104. Each MAP 1106 a-f can comprise a memory 1108 a-f and one or more field programmable gate arrays (FPGAs) 1110 a-f. The MAP provides a configurable functional unit and particular algorithms or portions of algorithms can be provided to the FPGAs 1110 a-f for processing in close coordination with a respective processor. For example, the MAPs can be used to evaluate algebraic expressions regarding the data model and to perform adaptive data restructuring in example instances. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use Direct Memory Access (DMA) to access an associated memory 1108 a-f, allowing it to execute tasks independently of, and asynchronously from the respective microprocessor 1102 a-f. In this configuration, a MAP can feed results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example instances, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some instances, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example instances, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.

In example instances, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other instances, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs) as referenced in FIG. 9, system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card, such as accelerator card 922 illustrated in FIG. 9.

The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Functionalization of a Device Surface

A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H₂SO₄ and 10% H₂O₂ for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N₂. The device was subsequently soaked in NH₄OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The device was then plasma cleaned by exposing the device surface to O₂. A SAMCO PC-300 instrument was used to plasma etch O₂ at 250 watts for 1 min in downstream mode.

The cleaned device surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface was resist coated using a Brewer Science 200X spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A descum process was used to remove residual resist using the SAMCO PC-300 instrument to O₂ plasma etch at 250 watts for 1 min.

The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with N₂. The functionalized surface was activated to serve as a support for polynucleotide synthesis.

Example 2: Synthesis of a 50-Mer Sequence on an Oligonucleotide Synthesis Device

A two dimensional oligonucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer″). The two-dimensional oligonucleotide synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to synthesize an exemplary polynucleotide of 50 bp (“50-mer polynucleotide”) using polynucleotide synthesis methods described herein.

The sequence of the 50-mer was as described in SEQ ID NO.: 48. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQ ID NO.: 48), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of oligos from the surface during deprotection.

The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 4 and an ABI synthesizer.

TABLE 4 Synthesis protocols General DNA Synthesis Table 4 Process Name Process Step Time (sec) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator to Flowcell 6 Activator Flow) Activator + 6 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Activator to Flowcell 0.5 Activator + 5 Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 DNA BASE ADDITION Activator Manifold Flush 2 (Phosphoramidite + Activator to Flowcell 5 Activator Flow) Activator + 18 Phosphoramidite to Flowcell Incubate for 25 sec 25 WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 CAPPING (CapA + B, 1:1, CapA + B to Flowcell 15 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) Acetonitrile to Flowcell 15 Acetonitrile System Flush 4 OXIDATION (Oxidizer Oxidizer to Flowcell 18 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 Acetonitrile System Flush 4 Acetonitrile to Flowcell 15 N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 23 N2 System Flush 4 Acetonitrile System Flush 4 DEBLOCKING (Deblock Deblock to Flowcell 36 Flow) WASH (Acetonitrile Wash Acetonitrile System Flush 4 Flow) N2 System Flush 4 Acetonitrile System Flush 4 Acetonitrile to Flowcell 18 N2 System Flush 4.13 Acetonitrile System Flush 4.13 Acetonitrile to Flowcell 15

The phosphoramidite/activator combination was delivered similar to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time.

The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M 12 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip.

Example 3: Synthesis of a 100-Mer Sequence on an Oligonucleotide Synthesis Device

The same process as described in Example 2 for the synthesis of the 50-mer sequence was used for the synthesis of a 100-mer polynucleotide (“100-mer polynucleotide”; 5′ CGGGATCCTTATCGTCATCGTCGTACAGATCCCGACCCATTTGCTGTCCACCAGTCATG CTAGCCATACCATGATGATGATGATGATGAGAACCCCGCAT##TTTTTTTTTT3′, where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes); SEQ ID NO.: 49) on two different silicon chips, the first one uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE and the second one functionalized with 5/95 mix of 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane, and the polynucleotides extracted from the surface were analyzed on a BioAnalyzer instrument.

All ten samples from the two chips were further PCR amplified using a forward (5′ATGCGGGGTTCTCATCATC3; SEQ ID NO.: 50) and a reverse (5′CGGGATCCTTATCGTCATCG3; SEQ ID NO.: 51) primer in a 50 uL PCR mix (25 uL NEB Q5 mastermix, 2.5 uL 10 uM Forward primer, 2.5 uL 10 uM Reverse primer, luL polynucleotide extracted from the surface, and water up to 50 uL) using the following thermalcycling program:

98° C., 30 sec

98° C., 10 sec; 63° C., 10 sec; 72° C., 10 sec; repeat 12 cycles

72° C., 2 min

The PCR products were also run on a BioAnalyzer, demonstrating sharp peaks at the 100-mer position. Next, the PCR amplified samples were cloned, and Sanger sequenced. Table 5 summarizes the results from the Sanger sequencing for samples taken from spots 1-5 from chip 1 and for samples taken from spots 6-10 from chip 2.

TABLE 5 Sequencing results Spot Error rate Cycle efficiency 1 1/763 bp 99.87% 2 1/824 bp 99.88% 3 1/780 bp 99.87% 4 1/429 bp 99.77% 5 1/1525 bp 99.93% 6 1/1615 bp 99.94% 7 1/531 bp 99.81% 8 1/1769 bp 99.94% 9 1/854 bp 99.88% 10 1/1451 bp 99.93%

Thus, the high quality and uniformity of the synthesized polynucleotides were repeated on two chips with different surface chemistries. Overall, 89% of the 100-mers that were sequenced were perfect sequences with no errors, corresponding to 233 out of 262.

Table 6 summarizes error characteristics for the sequences obtained from the polynucleotides samples from spots 1-10.

TABLE 6 Error characteristics Sample ID/Spot no. OSA_0046/1 OSA_0047/2 OSA_0048/3 OSA_0049/4 OSA_0050/5 Total Sequences 32 32 32 32 32 Sequencing Quality 25 of 28 27 of 27 26 of 30 21 of 23 25 of 26 Oligo Quality 23 of 25 25 of 27 22 of 26 18 of 21 24 of 25 ROI Match Count 2500 2698 2561 2122 2499 ROI Mutation 2 2 1 3 1 ROI Multi Base 0 0 0 0 0 Deletion ROI Small Insertion 1 0 0 0 0 ROI Single Base 0 0 0 0 0 Deletion Large Deletion Count 0 0 1 0 0 Mutation: G > A 2 2 1 2 1 Mutation: T > C 0 0 0 1 0 ROI Error Count 3 2 2 3 1 ROI Error Rate Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 in 834 in 1350 in 1282 in 708 in 2500 ROI Minus Primer MP MP MP MP MP Error Rate Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 in 763 in 824 in 780 in 429 in 1525 Sample ID/Spot no. OSA_0051/6 OSA_0052/7 OSA_0053/8 OSA_0054/9 OSA_0055/10 Total Sequences 32 32 32 32 32 Sequencing Quality 29 of 30 27 of 31 29 of 31 28 of 29 25 of 28 Oligo Quality 25 of 29 22 of 27 28 of 29 26 of 28 20 of 25 ROI Match Count 2666 2625 2899 2798 2348 ROI Mutation 0 2 1 2 1 ROI Multi Base 0 0 0 0 0 Deletion ROI Small Insertion 0 0 0 0 0 ROI Single Base 0 0 0 0 0 Deletion Large Deletion Count 1 1 0 0 0 Mutation: G > A 0 2 1 2 1 Mutation: T > C 0 0 0 0 0 ROI Error Count 1 3 1 2 1 ROI Error Rate Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 in 2667 in 876 in 2900 in 1400 in 2349 ROI Minus Primer MP MP MP MP MP Error Rate Err: ~1 Err: ~1 Err: ~1 Err: ~1 Err: ~1 in 1615 in 531 1769 in 854 1451

Example 4: Design of G Protein-Coupled Receptor Binding Domains Based on Conformational Ligand Interactions

G protein-coupled receptor (GPCR) binding domains were designed using interaction surfaces between conformational ligands that interact with GPCRs. Analysis of the interaction surfaces between chemokines and cytokines and the GPCRs indicated that the N-terminal peptide prior to the first conformational cysteine represents the activation peptide, and the core helical and beta-turn-beta topologies mediate interactions with the extracellular domain (ECD) of the GPCR.

An additional 254 GPCR ligands were designed based on cross-searching Uniprot and IUPHAR databases. The ligands represented 112 human, 71 rat, 4 pig, 1 sheep, and 1 cow derived interaction classes. The ligands were then collapsed to the following 101 cross-species ligand sequence annotations: ADM, ADM2, Agouti-related protein, Angiotensinogen, Annexin A1, Apelin, Apelin receptor early, Appetite regulating hormone, Beta-defensin 4A, C—C motif chemokine, C—X—C motif chemokine, Calcitonin, Calcitonin gene-related peptide, Cathepsin G, Cathepsin G (Fragment), Cholecystokinin, Complement C3, Complement C5, Complement C5 (Fragment), Corticoliberin, Cortistatin, Cytokine SCM-1 beta, Endothelin-2, Endothelin-3, Eotaxin, Fractalkine, Galanin peptides, Galanin-like peptide, Gastric inhibitory polypeptide, Gastrin, Gastrin-releasing peptide, Glucagon, Growth-regulated alpha protein, Heme-binding protein 1, Humanin, Insulin-like 3, Insulin-like peptide INSL5, Interleukin-8, Islet amyloid polypeptide, Kininogen-1, Lymphotactin, Metastasis-suppressor KiSS-1, Neurokinin-B, Neuromedin-B, Neuromedin-S, Neuromedin-U, Neuropeptide B, Neuropeptide S, Neuropeptide W, Neurotensin/neuromedin N, Orexigenic neuropeptide QRFP, Orexin, Oxytocin-neurophysin 1, Pancreatic prohormone, Parathyroid hormone, Parathyroid hormone-related protein, Peptide YY, Pituitary adenylate cyclase-activating, Platelet basic protein, Platelet factor 4, Prepronociceptin, Pro-FMRFamide-related neuropeptide FF, Pro-FMRFamide-related neuropeptide VF, Pro-MCH, Pro-neuropeptide Y, Pro-opiomelanocortin, Pro-thyrotropin releasing hormone, Proenkephalin-A, Proenkephalin-B, Progonadoliberin-1, Progonadoliberin-2, Prokineticin-1, Prokineticin-2, Prolactin-releasing peptide, Promotilin, Protachykinin-1, Protein Wnt-2, Protein Wnt-3a, Protein Wnt-4, Protein Wnt-5a, Protein Wnt-7b, Prothrombin, Proto-oncogene Wnt-1, Protooncogene Wnt-3, Putative uncharacterized protein, RCG55748, Retinoic acid receptor, Secretin, Somatoliberin, Somatostatin, Stromal cell-derived factor, T-kininogen 2, Tuberoinfundibular peptide of, Urocortin, Urocortin-2, Urocortin-3, Urotensin-2, Urotensin-2B, VEGF coregulated chemokine, VIP peptides, and Vasopressin-neurophysin 2-copeptin.

Structural analysis of the ligands was performed and indicated that a majority of them comprised the N-terminal activation peptide of about 11 amino acids. Motif variants were then created by trimming back the N-terminal activation peptide. As seen in Table 7, an exemplary set of variants were created based on the N-terminal activation peptide for stromal derived factor-1. The motif variants were also placed combinatorially at multiple positions in the CDR-H3. A total of 1016 motifs were extracted for placement in the CDR-H3. In addition, the motif variants were provided with variably boundary placement and with 5-20 substring variants that were also placed in the CDR-H3.

TABLE 7 Variant amino acid sequences for stromal derived factor-1 SEQ ID NO. Variant Amino Acid Sequence 53 1 ggggSDYKPVSLSYR 54 2 ggggDYKPVSLSYR 55 3 ggggYKPVSLSYR 56 4 ggggKPVSLSYR 57 5 ggggPVSLSYR

As seen in Table 8, an exemplary set of variants were created for interleukin-8 based on the following sequence:

(SEQ ID NO: 52) MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIK ELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.

TABLE 8 Variant amino acid sequences for inteleukin-8 SEQ ID NO. Variant Amino Acid Sequence 58 1 ggggSAALCEGAVLPRSA 59 2 ggggAALCEGAVLPRSA 60 3 ggggALCEGAVLPRSA 61 4 ggggLCEGAVLPRSA 62 5 ggggCEGAVLPRSA 63 6 ggggSAALCEGAVLPRSAKE 64 7 ggggAALCEGAVLPRSAKE 65 8 ggggALCEGAVLPRSAKE 66 9 ggggLCEGAVLPRSAKE 67 10 ggggCEGAVLPRSAKE 68 11 ggggSAALCEGAVLPRSAKELR 69 12 ggggAALCEGAVLPRSAKELR 70 13 ggggALCEGAVLPRSAKELR 71 14 ggggLCEGAVLPRSAKELR 72 15 ggggCEGAVLPRSAKELR

Example 5: Design of G Protein-Coupled Receptor Binding Domains Based on Peptide Ligand Interactions

GPCR binding domains were designed based on interaction surfaces between peptide ligands that interact with class B GPCRs. About 66 different ligands were used and include the following ligand sequence annotations: Adrenomedullin, Amylin, Angiotensin, Angiotensin I, Angiotensin II, Angiotensin III, Apelin, Apstatin, Big Endothelin, Big Gastrin, Bradykinin, Caerulein, Calcitonin, Calcitonin Gene Related Peptide, CGRP, Cholecystokinin, Endothelin, Endothelin 1, Endothelin 2, Endothelin 3, GIP, GIPs, GLP, Galanin, Gastrin, Ghrelin, Glucagon, IAPP, Kisspeptin, Mca, Metasti, Neuromedin, Neuromedin N, Neuropeptide, Neuropeptide F, Neuropeptide Y, Neurotensin, Nociceptin, Orexin, Orexin A, Orphanin, Oxytocin, Oxytocin Galanin, PACAP, PACAPs, PAR (Protease Activated Receptor) Peptides, PAR-1 Agonist, Pramlintide, Scyliorhinin I, Secretin, Senktide, Somatostatin, Somatostatin 14, Somatostatin 28, Substance P, Urotensin II, VIP, VIPs, Vasopressin, Xenin, cinnamoyl, furoyl, gastrin, holecystokinin, α-Mating Factor Pheromone. It was observed that the peptides formed a stabilized interaction with the GPCR extracellular domain (ECD).

Motif variants were generated based on the interaction surface of the peptides with the ECD as well as with the N-terminal GPCR ligand interaction surface. This was done using structural modeling. Exemplary motif variants were created based on glucagon like peptide's interaction with its GPCR as seen in Table 9. The motif variant sequences were generated using the following sequence from glucagon like peptide:

(SEQ ID NO: 73) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

TABLE 9 Variant amino acid sequences for glucagon like peptide SEQ ID NO. Variant Amino Acid Sequence 74 1 sggggsggggsggggHAEGTFTSDVSSYLEGQAAKEFIAWLV 75 2 sggggsggggsggggAEGTFTSDVSSYLEGQAAKEFIAWLV 76 3 sggggsggggsggggEGTFTSDVSSYLEGQAAKEFIAWLV 77 4 sggggsggggsggggGTFTSDVSSYLEGQAAKEFIAWLV 78 5 sggggsggggsggggTFTSDVSSYLEGQAAKEFIAWLV 79 6 sggggsggggsggggFTSDVSSYLEGQAAKEFIAWLV 80 7 sggggsggggsggggTSDVSSYLEGQAAKEFIAWLV 81 8 sggggsggggsggggSDVSSYLEGQAAKEFIAWLV 82 9 sggggsggggsggggDVSSYLEGQAAKEFIAWLV

Example 6: Design of G Protein-Coupled Receptor Binding Domains Based on Small Molecule Interactions

GPCR binding domains were designed based on interaction surfaces between small molecule ligands that interact with GPCRs. By analyzing multiple GPCR ligands, an amino acid library of Tyr, Pro, Phe, His, and Gly was designed as being able to recapitulate many of the structural contacts of these ligands. An exemplary motif variant that was generated based on these observations comprises the following sequence:

(SEQ ID NO: 83) sgggg(F,G,H,P,Y)(F,G,H,P,Y)(F,G,H,P,Y)(F,G,H,P,Y) (F,G,H,P,Y)(F,G,H,P,Y)(F,G,H,P,Y)(F,G,H,P,Y).

Example 7: Design of G Protein-Coupled Binding Domains Based on Extracellular Domain Interactions

GPCR binding domains were designed based on interaction surfaces on extracellular domains (ECDs) and extracellular loops (ECLs) of GPCRs. About 2,257 GPCRs from human (356), mouse (369), rat (259), cow (102), pig (60), primate, fish, fly, and over 200 other organisms were analyzed, and it was observed that ECDs provide multiple complementary contacts to other loops and helices of the GPCR at a length of 15 amino acids. Further analysis of the ECLs from the about 2,257 GPCRs and all solved structures of GPCRs demonstrated that the N-terminal ECD1 and ECL2 comprise longer extracellular sequences and provide GPCR extracellular contacts.

Motif variants were then generated based on these sequences. Exemplary variants based on the following sequence from retinoic acid induced protein 3 (GPRCSA) were generated:

(SEQ ID NO: 84) EYIVLTMNRTNVNVFSELSAPRRNED. See Table 10.

TABLE 10 Amino acid sequences SEQ ID NO. Variant Amino Acid Sequence 85 1 YIVLTMNRTNVNVFSELSAPRRNE 86 2 IVLTMNRTNVNVFSELSAPRRN 87 3 VLTMNRTNVNVFSELSAPRR

Example 8: Design of Antibody Scaffolds

To generate scaffolds, structural analysis, repertoire sequencing analysis of the heavy chain, and specific analysis of heterodimer high-throughput sequencing datasets were performed. Each heavy chain was associated with each light chain scaffold. Each heavy chain scaffold was assigned 5 different long CDR-H3 loop options. Each light chain scaffold was assigned 5 different L3 scaffolds. The heavy chain CDR-H3 stems were chosen from the frequently observed long H3 loop stems (10 amino acids on the N-terminus and the C-terminus) found both across individuals and across V-gene segments. The light chain scaffold L3s were chosen from heterodimers comprising long H3s. Direct heterodimers based on information from the Protein Data Bank (PDB) and deep sequencing datasets were used in which CDR H1, H2, L1, L2, L3, and CDR-H3 stems were fixed. The various scaffolds were then formatted for display on phage to assess for expression.

Structural Analysis

About 2,017 antibody structures were analyzed from which 22 structures with long CDR-H3s of at least 25 amino acids in length were observed. The heavy chains included the following: IGHV1-69, IGHV3-30, IGHV4-49, and IGHV3-21. The light chains identified included the following: IGLV3-21, IGKV3-11, IGKV2-28, IGKV1-5, IGLV1-51, IGLV1-44, and IGKV1-13. In the analysis, four heterodimer combinations were observed multiple times including: IGHV4-59/61-IGLV3-21, IGHV3-21-IGKV2-28, IGHV1-69-IGKV3-11, and IGHV1-69-IGKV1-5. An analysis of sequences and structures identified intra-CDR-H3 disulfide bonds in a few structures with packing of bulky side chains such as tyrosine in the stem providing support for long H3 stability. Secondary structures including beta-turn-beta sheets and a “hammerhead” subdomain were also observed.

Repertoire Analysis

A repertoire analysis was performed on 1,083,875 IgM+/CD27-naïve B cell receptor (BCR) sequences and 1,433,011 CD27+ sequences obtained by unbiased 5′RACE from 12 healthy controls. The 12 healthy controls comprised equal numbers of male and female and were made up of 4 Caucasian, 4 Asian, and 4 Hispanic individuals. The repertoire analysis demonstrated that less than 1% of the human repertoire comprises BCRs with CDR-H3s longer than 21 amino acids. A V-gene bias was observed in the long CDR3 subrepertoire, with IGHV1-69, IGHV4-34, IGHV1-18, and IGHV1-8 showing preferential enrichment in BCRs with long H3 loops. A bias against long loops was observed for IGHV3-23, IGHV4-59/61, IGHV5-51, IGHV3-48, IGHV3-53/66, IGHV3-15, IGHV3-74, IGHV3-73, IGHV3-72, and IGHV2-70. The IGHV4-34 scaffold was demonstrated to be autoreactive and had a short half-life.

Viable N-terminal and C-terminal CDR-H3 scaffold variation for long loops were also designed based on the 5′RACE reference repertoire. About 81,065 CDR-H3s of amino acid length 22 amino acids or greater were observed. By comparing across V-gene scaffolds, scaffold-specific H3 stem variation was avoided as to allow the scaffold diversity to be cloned into multiple scaffold references.

Heterodimer Analysis

Heterodimer analysis was performed on scaffolds having sequences as seen in FIGS. 12A-12C. Variant sequences and lengths of the scaffolds were assayed.

Structural Analysis

Structural analysis was performed using GPCR scaffolds of variant sequences and lengths were assayed. See FIG. 13.

Example 9: Generation of GPCR Antibody Libraries

Based on GPCR-ligand interaction surfaces and scaffold arrangements, libraries were designed and de novo synthesized. See Examples 4-8. Referring to FIG. 5, 10 variant sequences were designed for the variable domain, heavy chain 503, 237 variant sequences were designed for the heavy chain complementarity determining region 3 507, and 44 variant sequences were designed for the variable domain, light chain 513. The fragments were synthesized as three fragments as seen in FIG. 6 following similar methods as described in Examples 1-3.

Following de novo synthesis, 10 variant sequences were generated for the variable domain, heavy chain 602, 236 variant sequences were generated for the heavy chain complementarity determining region 3 604, and 43 variant sequences were designed for a region comprising the variable domain 606, light chain and CDR-L3 and of which 9 variants for variable domain, light chain were designed. This resulted in a library with about 10⁵ diversity (10×236×43). This was confirmed using next generation sequencing (NGS) with 16 million reads. As seen in FIG. 14, the normalized sequencing reads for each of the 10 variants for the variable domain, heavy chain was about 1. As seen in FIG. 15, the normalized sequencing reads for each of the 43 variants for the variable domain, light chain was about 1. As seen in FIG. 16, the normalized sequencing reads for 236 variant sequences for the heavy chain complementarity determining region 3 were about 1.

The various light and heavy chains were then tested for expression and protein folding. Referring to FIGS. 17A-17D, the 10 variant sequences for variable domain, heavy chain included the following: IGHV1-18, IGHV1-69, IGHV1-8 IGHV3-21, IGHV3-23, IGHV3-30/33m, IGHV3-28, IGHV3-74, IGHV4-39, and IGHV4-59/61. Of the 10 variant sequences, IGHV1-18, IGHV1-69, and IGHV3-30/33rn exhibited improved characteristics such as improved thermostability. Referring to FIGS. 18A-18F, 9 variant sequences for variable domain, light chain included the following: IGKV1-39, IGKV1-9, IGKV2-28, IGKV3-11, IGKV3-15, IGKV3-20, IGKV4-1, IGLV1-51, and IGLV2-14. Of the 9 variant sequences, IGKV1-39, IGKV3-15, IGLV1-51, and IGLV2-14 exhibited improved characteristics such as improved thermostability.

Example 10: Expression of GPCR Antibody Libraries in HEK293 Cells

Following generation of GPCR antibody libraries as in Example 13, about 47 GPCRs were selected for screening. GPCR constructs about 1.8 kb to about 4.5 kb in size were designed in a pCDNA3.1 vector. The GPCR constructs were then synthesized following similar methods as described in Examples 2-4 including hierarchal assembly. Of the 47 GPCR constructs, 46 GPCR constructs were synthesized.

The synthesized GPCR constructs were transfected in HEK293 and assayed for expression using immunofluorescence. Referring to FIGS. 19A-19C, HEK293 cells were transfected with the GPCR constructs comprising an N-terminally hemagglutinin (HA)-tagged human Y₁ receptor. Following 24-48 hours of transfection, cells were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. Cells were stained using fluorescent primary antibody directed towards the HA tag or secondary antibodies comprising a fluorophore and DAPI to visualize the nuclei in blue. Referring to FIGS. 19A-19C, human Y₁ receptor was visualized on the cell surface in non-permeabilized cells and on the cell surface and intracellularly in permeabilized cells.

GPCR constructs were also visualized by designing GPCR constructs comprising auto-fluorescent proteins. Referring to FIGS. 20A-20C, human Y₁ receptor comprised EYFP fused to its C-terminus, and human Y₅ receptor comprised ECFP fused to its C-terminus. HEK293 cells were transfected with human Y₁ receptor or co-transfected with human Y₁ receptor and human Y₅ receptor. Following transfection cells were washed and fixed with 4% paraformaldehyde. Cells were stained with DAPI. Localization of human Y₁ receptor and human Y₅ receptor were visualized by fluorescence microscopy.

Example 11: Design of Immunoglobulin Library

An immunoglobulin scaffold library was designed for placement of GPCR binding domains and for improving stability for a range of GPCR binding domain encoding sequences. The immunoglobulin scaffold included a VH domain attached with a VL domain with a linker. Variant nucleic acid sequences were generated for the framework elements and CDR elements of the VH domain and VL domain. The structure of the design is shown in FIG. 21A. A full domain architecture is shown in FIG. 21B. Sequences for the leader, linker, and pIII are listed in Table 11.

TABLE 11 Nucleotide sequences SEQ ID NO Domain Sequence 88 Leader GCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC 89 Linker GCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGC GGATCGCATGCATCC 90 pIII CGCGCGGCCGCTGGAAGCGGCTCCCACCATCACCATCACCAT

The VL domains that were designed include IGKV1-39, IGKV3-15, IGLV1-51, and IGLV2-14. Each of four VL domains were assembled with their respective invariant four framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (L1, L2, L3) elements. For IGKV1-39, there was 490 variants designed for L1, 420 variants designed for L2, and 824 variants designed for L3 resulting in a diversity of 1.7×10⁸ (490*420*824). For IGKV3-15, there was 490 variants designed for L1, 265 variants designed for L2, and 907 variants designed for L3 resulting in a diversity of 1.2×10⁸ (490*265*907). For IGLV1-51, there was 184 variants designed for L1, 151 variants designed for L2, and 824 variants designed for L3 resulting in a diversity of 2.3×10⁷ (184*151*824). IGLV2-14, 967 variants designed for L1, 535 variants designed for L2, and 922 variants designed for L3 resulting in a diversity of 4.8 10⁸ (967*535*922). Table 12 lists the amino acid sequences and nucleotide sequences for the four framework elements (FW1, FW2, FW3, FW4) for IGLV1-51. Table 13 lists the variable 3 CDR (L1, L2, L3) elements for IGLV1-51. Variant amino acid sequences and nucleotide sequences for the four framework elements (FW1, FW2, FW3, FW4) and the variable 3 CDR (L1, L2, L3) elements were also designed for IGKV1-39, IGKV3-15, and IGLV2-14.

TABLE 12 Sequences for IGLV1-51 framework elements SEQ SEQ ID ID Element NO Amino Acid Sequence NO Nucleotide Sequence IGLV1-51 FW1 91 QSVLTQPPSVSAAPGQKV 92 CAGTCTGTGTTGACGCAGCCGCCC TISC TCAGTGTCTGCGGCCCCAGGACAG AAGGTCACCATCTCCTGC FW2 93 WYQQLPGTAPKLLIY 94 TGGTATCAGCAGCTCCCAGGAACA GCCCCCAAACTCCTCATTTAT FW3 95 GIPDRFSGSKSGTSATLGI 96 GGGATTCCTGACCGATTCTCTGGC TGLQTGDEADYY TCCAAGTCTGGCACGTCAGCCACC CTGGGCATCACCGGACTCCAGACT GGGGACGAGGCCGATTATTAC FW4 97 GGGTKLTVL 98 GGCGGAGGGACCAAGCTGACCGTC CTA

TABLE 13 Sequences for IGLV1-51 CDR elements SEQ Amino Acid SEQ ID NO Sequence ID NO Nucleotide Sequence IGLV1-51-L1 99 SGSSSNIGS 282 TCTGGAAGCAGCTCCAACATTGGGAGTAATCATGTA NHVS TCC 100 SGSSSNIGN 283 TCTGGAAGCAGCTCCAACATTGGGAATAATTATCTA NYLS TCC 101 SGSSSNIAN 284 TCTGGAAGCAGCTCCAACATTGCGAATAATTATGTA NYVS TCC 102 SGSSPNIGN 285 TCTGGAAGCAGCCCCAACATTGGGAATAATTATGTA NYVS TCG 103 SGSRSNIGS 286 TCTGGAAGCAGATCCAATATTGGGAGTAATTATGTT NYVS TCG 104 SGSSSNVG 287 TCTGGAAGCAGCTCCAACGTTGGCGATAATTATGTT DNYVS TCC 105 SGSSSNIGIQ 288 TCTGGAAGCAGCTCCAACATTGGGATTCAATATGTA YVS TCC 106 SGSSSNVG 289 TCTGGAAGCAGCTCCAATGTTGGTAACAATTTTGTCT NNFVS CC 107 SGSASNIGN 290 TCTGGAAGCGCCTCCAACATTGGGAATAATTATGTA NYVS TCC 108 SGSGSNIGN 291 TCTGGAAGCGGCTCCAATATTGGGAATAATGATGTG NDVS TCC 109 SGSISNIGN 292 TCTGGAAGCATCTCCAACATTGGTAATAATTATGTA NYVS TCC 110 SGSISNIGK 293 TCTGGAAGCATCTCCAACATTGGGAAAAATTATGTG NYVS TCG 111 SGSSSNIGH 294 TCTGGAAGCAGCTCCAACATTGGGCATAATTATGTA NYVS TCG 112 PGSSSNIGN 295 CCTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 113 SGSTSNIGI 296 TCTGGAAGCACCTCCAACATTGGAATTCATTATGTA HYVS TCC 114 SGSSSNIGS 297 TCTGGAAGCAGCTCCAACATTGGCAGTCATTATGTT HYVS TCC 115 SGSSSNIGN 298 TCCGGAAGCAGCTCCAACATTGGAAATGAATATGTA EYVS TCC 116 SGSTSNIGN 299 TCTGGAAGCACCTCCAACATTGGAAATAATTATATA NYIS TCG 117 SGSSSNIGN 300 TCTGGAAGCAGCTCCAATATTGGGAATCATTTTGTA HFVS TCG 118 SGSSSNIGN 301 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTG NYVA GCC 119 SGSSSNIGS 302 TCTGGAAGCAGCTCCAACATTGGAAGTTATTATGTA YYVS TCC 120 SGSGFNIGN 303 TCTGGAAGTGGTTTCAACATTGGGAATAATTATGTC NYVS TCT 121 SGSTSNIGN 304 TCTGGAAGCACCTCCAACATTGGGAATAATTATGTG NYVS TCC 122 SGSSSDIGN 305 TCTGGAAGCAGCTCCGACATTGGCAATAATTATGTA NYVS TCC 123 SGSSSNIGN 306 TCTGGAAGCAGCTCCAACATTGGGAATAATGTTGTA NVVS TCC 124 SGSKSNIGK 307 TCTGGAAGCAAGTCTAACATTGGGAAAAATTATGTA NYVS TCC 125 SGSSTNIGN 308 TCTGGAAGCAGCACCAACATTGGGAATAATTATGTA NYVS TCC 126 SGSISNIGD 309 TCTGGAAGCATCTCCAACATTGGGGATAATTATGTA NYVS TCC 127 SGSSSNIGS 310 TCTGGAAGCAGCTCCAACATTGGGAGTAAGGATGTA KDVS TCA 128 SGSSSNIEN 311 TCTGGAAGCAGCTCCAACATTGAGAATAATGATGTA NDVS TCG 129 SGSSSNIGN 312 TCTGGAAGCAGCTCCAACATTGGGAATCATTATGTA HYVS TCC 130 SGSSSNIGK 313 TCTGGAAGCAGCTCCAACATTGGGAAGGATTTTGTC DFVS TCC 131 SGSTSNIGS 314 TCTGGCAGTACTTCCAACATCGGAAGTAATTTTGTTT NFVS CC 132 SGSTSNIGH 315 TCTGGAAGCACCTCCAACATTGGGCATAATTATGTA NYVS TCC 133 SASSSNIGN 316 TCTGCAAGCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 134 SGSSSSIGN 317 TCTGGAAGCAGCTCCAGCATTGGCAATAATTATGTA NYVS TCC 135 SGSSSTIGN 318 TCTGGAAGCAGCTCCACCATTGGGAATAATTATGTA NYVS TCC 136 SGSSSNIEN 319 TCTGGAAGCAGCTCCAACATTGAAAATAATTATGTA NYVS TCC 137 SGSSSNIGN 320 TCTGGAAGCAGCTCCAACATTGGGAATCAGTATGTA QYVS TCC 138 SGSSSNIGN 321 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVF TTC 139 SGSSSNIGR 322 TCTGGAAGCAGCTCCAACATTGGGAGGAATTATGTC NYVS TCC 140 SGGSSNIGN 323 TCTGGAGGCAGCTCCAACATTGGAAATTATTATGTA YYVS TCG 141 SGSSSNIGD 324 TCTGGAAGCAGCTCCAACATTGGAGATAATTATGTC NYVS TCC 142 SGGSSNIGI 325 TCTGGAGGCAGCTCCAACATTGGAATTAATTATGTA NYVS TCC 143 SGGSSNIGK 326 TCTGGAGGCAGCTCCAACATTGGGAAGAATTATGTA NYVS TCC 144 SGSSSNIGK 327 TCTGGAAGCAGCTCCAACATTGGGAAGAGATCTGTA RSVS TCG 145 SGSRSNIGN 328 TCTGGAAGCAGATCCAACATTGGGAATAACTATGTA NYVS TCC 146 SGSSSNIGN 329 TCGGGAAGCAGCTCCAACATTGGGAATAATCTTGTT NLVS TCC 147 SGSSSNIGIN 330 TCTGGAAGCAGCTCCAACATTGGGATCAATTATGTA YVS TCC 148 SGSSSNIGN 331 TCTGGAAGCAGCTCCAACATCGGGAATAATTTTGTA NFVS TCC 149 SGTSSNIGR 332 TCTGGAACCAGCTCCAACATTGGCAGAAATTTTGTA NFVS TCC 150 SGRRSNIGN 333 TCTGGAAGGAGGTCCAACATTGGAAATAATTATGTG NYVS TCC 151 SGGSFNIGN 334 TCTGGAGGCAGCTTCAATATTGGGAATAATTATGTA NYVS TCC 152 SGSTSNIGE 335 TCTGGAAGCACTTCCAACATTGGGGAGAATTATGTG NYVS TCC 153 SGSSSNIGS 336 TCTGGAAGCAGCTCCAATATTGGGAGTGATTATGTA DYVS TCC 154 SGTSSNIGS 337 TCTGGAACCAGCTCCAACATTGGGAGTAATTATGTA NYVS TCC 155 SGSSSNIGT 338 TCTGGAAGCAGCTCCAACATTGGGACTAATTTTGTA NFVS TCC 156 SGSSSNFGN 339 TCTGGAAGCAGCTCCAACTTTGGGAATAATTATGTA NYVS TCC 157 SGSTSNIGN 340 TCTGGAAGCACCTCCAACATTGGGAATAATCATGTA NHVS TCC 158 SGSSSNIGN 341 TCTGGAAGCAGCTCCAACATTGGGAATGATTTTGTA DFVS TCC 159 SGSSSDIGD 342 TCTGGAAGCAGCTCCGACATTGGCGATAATTATGTG NYVS TCC 160 SGSSSNIGK 343 TCTGGAAGCAGCTCCAACATTGGGAAATATTATGTA YYVS TCC 161 SGSSSNIGG 344 TCTGGAAGCAGCTCCAACATTGGCGGTAATTATGTA NYVS TCC 162 SGSSSNTGN 345 TCTGGAAGCAGCTCCAACACTGGGAATAATTATGTA NYVS TCC 163 SGSSSNVG 346 TCTGGAAGCAGCTCCAACGTTGGGAATAATTATGTG NNYVS TCT 164 SGSSSNIAN 347 TCTGGAAGCAGCTCCAACATTGCGAATAATTTTGTA NFVS TCC 165 SGSSSNIGN 348 TCTGGAAGCAGCTCCAACATTGGGAATGATTATGTA DYVS TCC 166 SGSTSNIEN 349 TCTGGAAGCACCTCCAATATTGAGAATAATTATGTT NYVS TCC 167 SGGSSNIGN 350 TCTGGAGGCAGCTCCAATATTGGCAATAATGATGTG NDVS TCC 168 SGSTSNIGN 351 TCTGGAAGCACCTCCAACATTGGGAATCATTATGTA HYVS TCC 169 SGSSSNIGD 352 TCAGGAAGCAGCTCCAATATTGGGGATAATGATGTA NDVS TCC 170 SGYSSNIGN 353 TCTGGATACAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 171 SGSGSNIGN 354 TCTGGAAGCGGCTCCAACATTGGAAATAATTTTGTA NFVS TCC 172 SGSSSNIWN 355 TCTGGAAGCAGCTCCAACATTTGGAATAATTATGTA NYVS TCC 173 FGSSSNIGN 356 TTTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 174 SGSSSNIEK 357 TCTGGAAGCAGCTCCAACATTGAGAAGAATTATGTA NYVS TCC 175 SGSRSNIGN 358 TCTGGAAGTAGATCCAATATTGGAAATTATTATGTA YYVS TCC 176 SGTKSNIGN 359 TCTGGAACCAAGTCAAACATTGGGAATAATTATGTA NYVS TCT 177 SGSTSNIGN 360 TCTGGAAGCACCTCCAACATTGGGAATTATTATGTA YYVS TCC 178 SGTSSNIGN 361 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTG NYVA GCC 179 PGTSSNIGN 362 CCTGGAACCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 180 SGSTSNIGI 363 TCCGGAAGCACCTCCAACATTGGGATTAATTATGTA NYVS TCC 181 SGSSSNIGS 364 TCTGGAAGCAGCTCCAACATTGGGAGTAATCTGGTA NLVS TCC 182 SGSSSNIEN 365 TCTGGAAGCAGCTCCAACATTGAGAATAATCATGTA NHVS TCC 183 SGTRSNIGN 366 TCTGGAACCAGGTCCAACATCGGCAATAATTATGTT NYVS TCG 184 SGSTSNIGD 367 TCTGGAAGCACCTCCAACATTGGGGACAATTATGTT NYVS TCC 185 SGGSSNIGK 368 TCTGGAGGCAGTTCCAACATTGGGAAGAATTTTGTA NFVS TCC 186 SGSRSDIGN 369 TCTGGAAGCAGGTCCGACATTGGGAATAATTATGTA NYVS TCC 187 SGTSSNIGN 370 TCTGGAACTAGCTCCAACATTGGGAATAATGATGTA NDVS TCC 188 SGSSSNIGS 371 TCTGGAAGCAGCTCCAACATTGGGAGTAAATATGTA KYVS TCA 189 SGSSFNIGN 372 TCTGGAAGCAGCTTCAACATTGGGAATAATTATGTA NYVS TCC 190 SGSSSNIGN 373 TCTGGAAGCAGCTCCAACATTGGGAATACTTATGTA TYVS TCC 191 SGSSSNIGD 374 TCTGGAAGCAGCTCCAATATTGGGGATAATCATGTA NHVS TCC 192 SGSSSNIGN 375 TCTGGAAGCAGCTCCAACATTGGCAATAATCATGTT NHVS TCC 193 SGSTSNIGN 376 TCTGGAAGCACCTCCAACATTGGGAATAATGATGTA NDVS TCC 194 SGSRSNVG 377 TCTGGAAGCAGATCCAACGTTGGCAATAATTATGTT NNYVS TCA 195 SGGTSNIGK 378 TCCGGAGGCACCTCCAACATTGGGAAGAATTATGTG NYVS TCT 196 SGSSSNIAD 379 TCTGGAAGCAGCTCCAACATTGCCGATAATTATGTT NYVS TCC 197 SGSSSNIGA 380 TCTGGAAGCAGCTCCAACATTGGCGCCAATTATGTA NYVS TCC 198 SGSSSNIGS 381 TCTGGAAGCAGCTCCAACATTGGGAGTAATTATGTG NYVA GCC 199 SGSSSNIGN 382 TCTGGAAGCAGCTCCAACATTGGGAACAATTTTCTC NFLS TCC 200 SGRSSNIGK 383 TCTGGAAGAAGCTCCAACATTGGGAAGAATTATGTA NYVS TCC 201 SGSSPNIGA 384 TCTGGAAGCAGCCCCAACATTGGGGCTAATTATGTA NYVS TCC 202 SGSSSNIGP 385 TCCGGAAGCAGCTCCAACATTGGGCCTAATTATGTG NYVS TCC 203 SGSSSTIGN 386 TCTGGAAGCAGCTCCACCATTGGGAATAATTATATA NYIS TCC 204 SGSSSNIGN 387 TCTGGAAGCAGCTCCAACATTGGGAATTATTTTGTA YFVS TCC 205 SGSRSNIGN 388 TCTGGAAGCCGCTCCAACATTGGTAATAATTTTGTAT NFVS CC 206 SGGSSNIGS 389 TCTGGAGGCAGCTCCAACATTGGGAGTAATTTTGTA NFVS TCC 207 SGSSSNIGY 390 TCTGGAAGCAGCTCCAACATTGGGTATAATTATGTA NYVS TCC 208 SGTSSNIEN 391 TCTGGAACCAGCTCGAACATTGAGAACAATTATGTA NYVS TCC 209 SGSSSNIGN 392 TCTGGAAGTAGCTCCAACATTGGGAATTATTATGTA YYVS TCC 210 SGSTSNIGK 393 TCTGGAAGCACCTCCAACATTGGGAAGAATTATGTA NYVS TCC 211 SGSSSNIGT 394 TCTGGAAGCAGTTCCAACATTGGGACTTATTATGTCT YYVS CT 212 SGSSSNVG 395 TCTGGAAGCAGCTCCAACGTTGGGAAAAATTATGTA KNYVS TCT 213 SGSTSNIGD 396 TCTGGAAGCACCTCCAACATTGGGGATAATTTTGTA NFVS TCC 214 SGSTSNIGT 397 TCTGGAAGCACCTCCAACATTGGAACTAATTATGTT NYVS TCC 215 SGGTSNIGN 398 TCTGGAGGTACTTCCAACATTGGGAATAATTATGTC NYVS TCC 216 SGSYSNIGN 399 TCTGGAAGCTACTCCAATATTGGGAATAATTATGTA NYVS TCC 217 SGSSSNIED 400 TCTGGAAGCAGCTCCAACATTGAAGATAATTATGTA NYVS TCC 218 SGSSSNIGK 401 TCTGGAAGCAGCTCCAACATTGGGAAACATTATGTA HYVS TCC 219 SGSGSNIGS 402 TCCGGTTCCGGCTCAAACATTGGAAGTAATTATGTC NYVS TCC 220 SGSSSNIGN 403 TCTGGAAGCAGCTCCAACATTGGAAATAATTATATA NYIS TCA 221 SGASSNIGN 404 TCTGGAGCCAGTTCCAACATTGGGAATAATTATGTT NYVS TCC 222 SGRTSNIGN 405 TCTGGACGCACCTCCAACATCGGGAACAATTATGTA NYVS TCC 223 SGGSSNIGS 406 TCTGGAGGCAGCTCCAATATTGGGAGTAATTACGTA NYVS TCC 224 SGSGSNIGN 407 TCTGGAAGCGGCTCCAACATTGGGAATAATTATGTA NYVS TCC 225 SGSTSNIGS 408 TCTGGAAGCACCTCCAACATTGGGAGTAATTATGTA NYVS TCC 226 SGSSSSIGN 409 TCTGGAAGCAGCTCCAGCATTGGGAATAATTATGTG NYVA GCG 227 SGSSSNLGN 410 TCTGGAAGCAGTTCCAACCTTGGAAATAATTATGTA NYVS TCC 228 SGTSSNIGK 411 TCTGGAACCAGCTCCAACATTGGGAAAAATTATGTA NYVS TCC 229 SGSSSDIGN 412 TCTGGAAGCAGCTCCGATATTGGGAACAAGTATATA KYIS TCC 230 SGSSSNIGS 413 TCTGGAAGCAGCTCCAACATTGGAAGTAATTACATA NYIS TCC 231 SGSTSNIGA 414 TCTGGAAGCACCTCCAACATTGGGGCTAACTATGTG NYVS TCC 232 SGSSSNIGN 415 TCTGGAAGCAGCTCCAACATTGGGAATAAGTATGTA KYVS TCC 233 SGSSSNIGN 416 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGGA NYGS TCC 234 SGSTSNIAN 417 TCTGGAAGCACCTCCAACATTGCGAATAATTATGTA NYVS TCC 235 SGSYSNIGS 418 TCTGGAAGCTACTCCAATATTGGGAGTAATTATGTA NYVS TCC 236 SGSSSNIGS 419 TCTGGAAGCAGCTCCAACATTGGGAGTAATTTTGTA NFVS TCC 237 SGSSSNLEN 420 TCTGGAAGCAGCTCCAATCTTGAGAATAATTATGTA NYVS TCC 238 SGSISNIGSN 421 TCTGGAAGCATCTCCAATATTGGCAGTAATTATGTA YVS TCC 239 SGSSSDIGS 422 TCTGGAAGCAGCTCCGACATTGGGAGTAATTATGTA NYVS TCC 240 SGSSSNIGT 423 TCTGGAAGCAGCTCCAACATTGGGACTAATTATGTA NYVS TCC 241 SGSSSNIGK 424 TCTGGAAGCAGCTCCAACATTGGGAAGAATTTTGTA NFVS TCC 242 SGSSSNIGN 425 TCTGGAAGCAGCTCCAACATTGGGAATAATTTTATA NFIS TCC 243 SGGSSNIGN 426 TCTGGAGGCAGCTCCAACATTGGCAATAATTATGTT NYVS TCC 244 SGSSSNIGE 427 TCTGGAAGCAGCTCCAACATTGGGGAGAATTATGTA NYVS TCC 245 SGSSSNIGN 428 TCTGGAAGCAGCTCCAATATTGGGAATAATTTTGTG NFVA GCC 246 SGGSSNIGN 429 TCTGGAGGCAGCTCCAACATTGGGAATAATTATGTA NYVA GCC 247 SGSSSHIGN 430 TCTGGAAGCAGCTCCCACATTGGAAATAATTATGTA NYVS TCC 248 SGSSSNIGS 431 TCTGGAAGCAGCTCCAATATTGGAAGTAATGATGTA NDVS TCG 249 SGSSSNIGN 432 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVT ACC 250 SGSSSNIGN 433 TCTGGAAGCAGCTCCAACATTGGGAATAATCCTGTA NPVS TCC 251 SGGSSNIGN 434 TCTGGAGGCAGCTCCAATATTGGGAATCATTATGTA HYVS TCC 252 SGTSSNIGN 435 TCTGGAACCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 253 SGSSSNIGS 436 TCTGGAAGCAGCTCCAACATTGGAAGTAATTATGTC NYVS TCG 254 SGGTSNIGS 437 TCTGGAGGCACCTCCAACATTGGAAGTAATTATGTA NYVS TCC 255 SGSKSNIGN 438 TCTGGAAGCAAGTCCAACATTGGGAATAATTATGTA NYVS TCC 256 SGRSSNIGN 439 TCTGGAAGAAGCTCCAACATTGGGAATAATTATGTA NYVS TCG 257 SGSSSNVGS 440 TCTGGAAGCAGCTCCAACGTTGGGAGTAATTATGTT NYVS TCC 258 SGSTSNIGN 441 TCTGGAAGCACCTCCAATATTGGGAATAATTTTGTA NFVS TCC 259 SGSNFNIGN 442 TCTGGAAGCAACTTCAACATTGGGAATAATTATGTC NYVS TCC 260 SGSTSNIGY 443 TCTGGAAGCACCTCCAATATTGGATATAATTATGTA NYVS TCC 261 SGSSSNIVS 444 TCTGGAAGCAGCTCCAATATTGTAAGTAATTATGTA NYVS TCC 262 SGTSSNIGN 445 TCTGGAACCAGCTCCAACATTGGGAATAATTTTGTA NFVS TCC 263 SGSSSNIGR 446 TCTGGAAGCAGCTCCAACATTGGGAGGAATTTTGTG NFVS TCC 264 SGTTSNIGN 447 TCTGGAACGACCTCCAACATTGGGAATAATTATGTC NYVS TCC 265 SGSSSNIGN 448 TCTGGAAGCAGCTCCAACATTGGGAATAATGATGTA NDVS TCC 266 SGSSSNIGN 449 TCTGGAAGCAGCTCCAACATTGGGAATCATGATGTA HDVS TCC 267 SGSSSNIGS 450 TCTGGAAGCAGCTCCAACATTGGAAGTAGTCATGTA SHVS TCC 268 SGSSSNIGIH 451 TCTGGAAGCAGCTCCAACATTGGGATTCATTATGTA YVS TCC 269 SGGGSNIGY 452 TCTGGAGGCGGCTCCAACATTGGCTATAATTATGTC NYVS TCC 270 SGSSSNIGD 453 TCTGGAAGCAGCTCCAACATTGGGGATCATTATGTG HYVS TCG 271 SGSSSNLGK 454 TCTGGAAGCAGCTCCAACCTTGGGAAGAATTATGTA NYVS TCT 272 SGSSSNIGD 455 TCTGGAAGCAGCTCCAACATTGGCGATAATTTTGTA NFVS TCC 273 SGSTSNIEK 456 TCTGGAAGCACCTCCAACATTGAGAAAAACTATGTA NYVS TCG 274 SGSSSNIGK 457 TCTGGAAGCAGCTCCAACATTGGGAAGGATTATGTA DYVS TCC 275 SGSSSNIGK 458 TCTGGAAGCAGCTCCAACATTGGGAAGAATTATGTA NYVS TCC 276 SGSSSNIGN 459 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 277 SGSSSNIGN 460 TCTGGAAGCAGCTCCAACATTGGGAATAATTATGCC NYAS TCC 278 SGISSNIGN 461 TCTGGAATCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 279 TGSSSNIGN 462 ACTGGAAGCAGCTCCAACATTGGGAATAATTATGTA NYVS TCC 280 SGTSSNIGN 463 TCTGGAACCAGCTCCAACATTGGGAATAATCATGTT NHVS TCC 281 SGSRSNIGK 464 TCTGGAAGTCGTTCCAACATTGGGAAAAATTATGTA NYVS TCC IGLV1-51-L2 465 DNNKRPP 616 GACAATAATAAGCGACCCCCA 466 ENNRRPS 617 GAGAATAATAGGCGACCCTCA 467 DNNKQPS 618 GACAATAATAAGCAACCCTCA 468 DNNKRPL 619 GACAATAACAAGCGACCCTTG 469 DNDKRPA 620 GACAATGATAAGCGACCCGCA 470 DNHERPS 621 GACAATCATGAGCGACCCTCA 471 ENRKRPS 622 GAAAACCGTAAGCGACCCTCA 472 DNDQRPS 623 GACAATGATCAGCGACCCTCA 473 ENYKRPS 624 GAGAATTATAAGCGACCCTCA 474 ENTKRPS 625 GAAAATACTAAGCGACCCTCA 475 DTEKRPS 626 GACACTGAGAAGAGGCCCTCA 476 DNDKRPP 627 GACAATGATAAGCGACCCCCA 477 DHNKRPS 628 GACCATAATAAGCGACCCTCA 478 GNNERPS 629 GGCAATAATGAGCGACCCTCA 479 DTSKRPS 630 GACACTAGTAAGCGACCCTCA 480 EYNKRPS 631 GAATATAATAAGCGCCCCTCA 481 ENIKRPS 632 GAAAATATTAAGCGACCCTCA 482 DNVKRPS 633 GACAATGTTAAGCGACCCTCA 483 ENDKRSS 634 GAAAACGATAAACGATCCTCA 484 ENNKRHS 635 GAAAATAATAAGCGACACTCA 485 GNDQRPS 636 GGAAATGATCAGCGACCCTCA 486 DNDRRPS 637 GACAATGATAGGCGACCCTCA 487 DNHKRPS 638 GACAATCATAAGCGGCCCTCA 488 DNNDRPS 639 GACAATAATGACCGACCCTCA 489 ENNQRPS 640 GAGAATAATCAGCGACCCTCA 490 DNNQRPS 641 GACAATAATCAGCGACCCTCA 491 ENVKRPS 642 GAGAATGTTAAGCGACCCTCA 492 DTYKRPS 643 GACACTTATAAGAGACCCTCA 493 NNNNRPS 644 AACAATAATAACCGACCCTCA 494 GNNNRPS 645 GGCAATAATAATCGACCCTCA 495 ENDQRPS 646 GAAAATGATCAGCGACCCTCA 496 DNNKRAS 647 GACAATAATAAGCGAGCCTCA 497 DNDKRPL 648 GACAATGATAAGCGACCCTTA 498 DTDERPS 649 GACACTGATGAGCGACCTTCA 499 DNRKRPS 650 GACAATAGGAAGCGACCCTCA 500 DNDARPS 651 GACAATGATGCTCGACCCTCA 501 DNNKRLS 652 GACAATAATAAGCGACTCTCA 502 DNDKRAS 653 GACAATGATAAGCGAGCCTCA 503 DNTERPS 654 GACAATACTGAGCGACCCTCA 504 DNNIRPS 655 GACAATAATATTCGACCCTCA 505 DNKRRPS 656 GACAATAAGAGGCGACCCTCA 506 DDNNRPS 657 GACGATAATAACCGACCCTCA 507 ANNRRPS 658 GCGAATAATCGACGACCCTCA 508 DNDKRLS 659 GACAATGATAAGCGACTGTCA 509 DNNKRPA 660 GACAATAATAAGCGACCCGCA 510 DNYRRPS 661 GACAATTATAGACGTCCCTCA 511 ANDQRPS 662 GCCAATGATCAGCGACCCTCA 512 DNDKRRS 663 GACAATGATAAGCGACGCTCA 513 DKNERPS 664 GACAAGAATGAGCGACCCTCA 514 DNKERPS 665 GACAATAAGGAGCGACCCTCA 515 DNNKGPS 666 GACAATAATAAGGGACCCTCA 516 ENDRRPS 667 GAAAATGATAGACGACCCTCA 517 ENDERPS 668 GAAAATGATGAGCGACCCTCA 518 QNNKRPS 669 CAAAATAATAAGCGACCCTCA 519 DNRERPS 670 GACAATCGTGAGCGACCCTCA 520 DNNRRPS 671 GACAATAATAGACGACCCTCA 521 GNNRRPS 672 GGAAATAATAGGCGACCCTCA 522 DNDNRPS 673 GACAATGATAACCGACCCTCA 523 EDNKRPS 674 GAAGATAATAAGCGACCCTCA 524 DDDERPS 675 GACGATGATGAGCGGCCCTCA 525 ASNKRPS 676 GCAAGTAATAAGCGACCCTCA 526 DNNKRSS 677 GACAATAATAAGCGATCCTCA 527 QNNERPS 678 CAAAATAATGAGCGACCCTCA 528 DDDRRPS 679 GACGATGATAGGCGACCCTCA 529 NNDKRPS 680 AACAATGATAAGCGACCCTCA 530 DNNNRPS 681 GACAATAATAACCGACCCTCA 531 DNNVRPS 682 GACAATAATGTGCGACCCTCA 532 ENNERPS 683 GAAAATAATGAGCGACCCTCA 533 DNNHRPS 684 GACAATAATCACCGACCCTCA 534 DNDERPS 685 GACAATGATGAGCGCCCCTCG 535 DNIRRPS 686 GACAATATCCGGCGACCCTCA 536 DFNKRPS 687 GACTTTAATAAGCGACCCTCA 537 ETNKRPS 688 GAAACTAATAAGCGACCCTCA 538 NDNKRPS 689 AACGATAATAAGCGACCCTCA 539 DDNKRPS 690 GACGATAATAAGCGACCCTCA 540 DNYKRPS 691 GACAATTATAAGCGACCCTCA 541 HNNKRPS 692 CACAATAATAAGCGACCCTCA 542 DNHQRPS 693 GACAATCATCAGCGACCCTCA 543 DNYKRAS 694 GACAATTATAAGCGAGCCTCA 544 DNIKRPS 695 GACAATATTAAGCGACCCTCA 545 DTHKRPS 696 GACACTCATAAGCGACCCTCA 546 DTNRRPS 697 GACACTAATAGGCGACCCTCT 547 DTNQRPS 698 GACACTAATCAGCGACCCTCA 548 ESDKRPS 699 GAAAGTGATAAGCGACCCTCA 549 DNDKRSS 700 GACAATGATAAGCGATCTTCG 550 GSNKRPS 701 GGCAGTAATAAGCGACCCTCA 551 DNNKRVS 702 GACAATAACAAGCGAGTTTCA 552 NNNRRPS 703 AACAATAATAGGCGACCCTCA 553 DNFKRPS 704 GACAATTTTAAGCGACCCTCA 554 ENDKRPS 705 GAAAATGATAAACGACCCTCA 555 ENNKRLS 706 GAAAATAATAAGCGACTCTCA 556 ADNKRPS 707 GCAGATAATAAGCGACCCTCA 557 EDNERPS 708 GAAGATAATGAGCGCCCCTCA 558 DTDQRPS 709 GACACTGATCAGCGACCCTCA 559 DNYQRPS 710 GACAATTATCAGCGACCCTCA 560 DENKRPS 711 GACGAGAATAAGCGACCCTCA 561 DTNKRPS 712 GACACTAATAAGCGACCCTCA 562 DDYRRPS 713 GACGATTATCGGCGACCCTCA 563 DNDKRHS 714 GACAACGATAAGCGGCACTCA 564 ENDNRPS 715 GAAAATGATAATCGACCCTCA 565 DDNERPS 716 GACGATAATGAGCGCCCCTCA 566 DNKKRPS 717 GACAATAAGAAGCGACCCTCA 567 DVDKRPS 718 GACGTTGATAAGCGACCCTCA 568 ENKKRPS 719 GAAAATAAAAAACGACCCTCT 569 VNDKRPS 720 GTCAATGATAAGCGACCCTCA 570 DNDHRPS 721 GACAATGATCACCGACCCTCA 571 DINKRPS 722 GACATTAATAAGCGACCCTCA 572 ANNERPS 723 GCCAATAATGAGCGACCCTCA 573 DNENRPS 724 GACAATGAAAACCGACCGTCA 574 GDDKRPS 725 GGCGATGATAAGCGACCCTCA 575 ANNQRPS 726 GCCAATAATCAGCGACCTTCA 576 DDDKRPS 727 GACGATGATAAGCGACCCTCA 577 YNNKRPS 728 TACAATAATAAGCGGCCCTCA 578 EDDKRPS 729 GAAGATGATAAGCGACCCTCA 579 ENNNRPS 730 GAAAACAATAACCGACCCTCG 580 DNNLRPS 731 GACAATAATCTGCGACCCTCA 581 ESNKRPS 732 GAGAGTAACAAGCGACCCTCA 582 DTDKRPS 733 GACACTGATAAGCGGCCCTCA 583 DDDQRPS 734 GACGATGATCAGCGACCCTCA 584 VNNKRPS 735 GTGAATAATAAGAGACCCTCC 585 DDYKRPS 736 GACGATTATAAGCGACCCTCA 586 DNTKRPS 737 GACAATACTAAGCGACCCTCA 587 DDTERPS 738 GACGATACTGAGCGACCCTCA 588 GNDKRPS 739 GGCAATGATAAGCGACCCTCA 589 DNEKRPS 740 GACAATGAAAAGCGACCCTCA 590 DNDDRPS 741 GACAATGATGACCGACCCTCA 591 DDNRRPS 742 GACGATAATAGGCGTCCCTCA 592 GNNKRPS 743 GGCAATAATAAGCGACCCTCA 593 ANDKRPS 744 GCCAATGATAAGCGACCCTCA 594 DNNKRHS 745 GACAATAATAAGCGACACTCA 595 DDNQRPS 746 GACGACAATCAGCGACCCTCA 596 GNDRRPS 747 GGCAATGATAGGCGACCCTCA 597 DNHNRPS 748 GACAATCATAACCGACCCTCA 598 DNYERPS 749 GACAATTATGAGCGACCCTCA 599 ENNKRSS 750 GAAAATAATAAGCGATCCTCA 600 DDHKRPS 751 GACGATCATAAGCGGCCCTCA 601 DNNKRRS 752 GACAATAATAAACGACGTTCA 602 DNDKRPS 753 GACAATGATAAGCGACCGTCA 603 DKNKRPS 754 GACAAGAATAAGCGACCCTCA 604 DNNKRPS 755 GACAATAATAAGCGACCCTCA 605 DIDKRPS 756 GACATTGATAAGCGACCCTCA 606 DDKKRPS 757 GACGATAAGAAGCGACCCTCA 607 ANNKRPS 758 GCCAATAATAAGCGACCCTCA 608 DNDKGPS 759 GACAATGATAAGGGACCCTCA 609 EDNRRPS 760 GAAGATAATAGGCGACCCTCA 610 ENNKRPS 761 GAGAATAATAAGCGACCCTCA 611 NNNKRPS 762 AACAATAATAAGCGACCCTCA 612 DNNERPS 763 GACAATAATGAGCGACCCTCA 613 DNIQRPS 764 GACAATATTCAGCGACCCTCA 614 DNNYRPS 765 GACAATAATTACCGACCCTCA 615 DNYNRPS 766 GACAATTATAACCGACCCTCA IGLV1-51-L3 767 CGTWDTSL 1591 TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTG SAVVF TTC 768 CGTWDTSL 1592 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGTG SAGVF TTC 769 CGTWDTSL 1593 TGCGGAACATGGGATACCAGCCTGAGTGCTTGGGTG SAWVF TTC 770 CGTWDRSL 1594 TGCGGAACATGGGATAGGAGCCTGAGTGCGGGGGT SAGVF GTTC 771 CGTWDRSL 1595 TGCGGAACATGGGATAGGAGCCTGAGTGCTTGGGTA SAWVF TTT 772 CGTWDTSL 1596 TGCGGAACATGGGATACCAGCCTGAGTGGTGGGGTG SGGVF TTC 773 CGTWDTSL 1597 TGCGGAACATGGGATACTAGCCTGCGTGCTGGCGTC RAGVF TTC 774 CGTWDRSL 1598 TGCGGAACATGGGATAGGAGCCTGAGTGTTTGGGTG SVWVF TTC 775 CGTWDTSL 1599 TGCGGAACATGGGATACCAGTCTGAGTGTTGTGGTC SVVVF TTC 776 CGTWDTSL 1600 TGCGGAACGTGGGATACCAGCCTGAGTGCTGCGGTG SAAVF TTC 777 CGAWDTSL 1601 TGCGGAGCATGGGATACCAGCCTGAGTGCTGGAGTG SAGVF TTC 778 CATWDTSL 1602 TGCGCAACATGGGATACCAGCCTGAGTGCTGTGGTA SAVVF TTC 779 CATWDTSL 1603 TGCGCAACATGGGATACCAGCCTGAGTGCTGGTGTG SAGVF TTC 780 CGTWESSL 1604 TGTGGAACATGGGAGAGCAGCCTGAGTGCTTGGGTG SAWVF TTC 781 CGTWDTTL 1605 TGCGGAACATGGGATACCACCCTGAGTGCGGGTGTC SAGVF TTC 782 CGTWDTSL 1606 TGCGGAACATGGGATACTAGCCTGAGTGTGTGGGTG SVWVF TTC 783 CGTWDTSL 1607 TGCGGAACATGGGATACTAGCCTGAGTGTTGGGGTG SVGVF TTC 784 CGTWDTSL 1608 TGCGGAACATGGGACACCAGTCTGAGCACTGGCGTC STGVF TTC 785 CGTWDTSL 1609 TGCGGAACATGGGATACCAGCCTGAGTGGTGTGGTC SGVVF TTC 786 CGTWDTSL 1610 TGCGGAACATGGGATACCAGCCTGAGTGCTTATGTC SAYVF TTC 787 CGTWDTSL 1611 TGCGGAACATGGGATACCAGCCTGAGTGCTGAGGTG SAEVF TTC 788 CGTWDTGL 1612 TGCGGAACATGGGATACCGGCCTGAGTGCTGGGGTA SAGVF TTC 789 CGTWDRSL 1613 TGCGGAACGTGGGATAGGAGCCTGAGTGCTTATGTC SAYVF TTC 790 CGTWDRSL 1614 TGCGGAACATGGGATAGGAGCCTCAGTGCCGTGGTA SAVVF TTC 791 CGTWDNTL 1615 TGCGGAACATGGGATAACACCCTGAGTGCGTGGGTG SAWVF TTC 792 CGTWDNRL 1616 TGCGGAACATGGGATAACAGGCTGAGTGCTGGGGT SAGVF GTTC 793 CGTWDISLS 1617 TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGTG AWVF TTC 794 CGTWHSSL 1618 TGCGGAACATGGCATAGCAGCCTGAGTGCTGGGGTA SAGVF TTC 795 CGTWGSSL 1619 TGCGGAACATGGGGTAGCAGTTTGAGTGCTTGGGTG SAWVF TTC 796 CGTWESSL 1620 TGCGGAACATGGGAGAGCAGCCTGAGTGGTTGGGT SGWVF GTTC 797 CGTWESSL 1621 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTGGTT SAVVF TTC 798 CGTWDYSL 1622 TGCGGAACATGGGATTACAGCCTGAGTGCTGTGGTA SAVVF TTC 799 CGTWDYSL 1623 TGCGGAACATGGGATTACAGCCTGAGTGCTGGGGTA SAGVF TTC 800 CGTWDVSL 1624 TGCGGAACATGGGATGTCAGCCTGAGTGTTGGAGTG SVGVF TTC 801 CGTWDTTL 1625 TGCGGAACATGGGATACCACCCTGAGTGCTGTGGTT SAVVF TTC 802 CGTWDTTL 1626 TGCGGAACATGGGATACCACTCTGAATATTGGGGTG NIGVF TTC 803 CGTWDTSL 1627 TGCGGAACATGGGATACCAGCCTGACTGCTGTGGTA TAVVF TTC 804 CGTWDTSL 1628 TGCGGAACCTGGGATACCAGCCTGACTGCTGCTGTG TAAVF TTC 805 CGTWDTSL 1629 TGCGGCACATGGGATACCAGCCTGAGTGTGGGGCTA SVGLF TTC 806 CGTWDTSL 1630 TGCGGAACCTGGGATACCAGCCTGAGTGGTAGGGTG SGRVF TTC 807 CGTWDTSL 1631 TGCGGAACATGGGATACCAGCCTGAGTGGTGCAGTG SGAVF TTC 808 CGTWDTSL 1632 TGCGGAACATGGGATACCAGCCTGAGTGCTGGCCTG SAGLF TTC 809 CGTWDTSL 1633 TGCGGAACATGGGATACCAGCCTGAGTGCTGGAGG SAGGVF GGTCTTC 810 CGTWDTSL 1634 TGCGGAACATGGGATACCAGCCTGCGTGCTTATGTC RAYVF TTC 811 CGTWDTSL 1635 TGCGGAACATGGGATACTAGTTTGCGTGCTTGGGTA RAWVF TTC 812 CGTWDTSL 1636 TGCGGAACATGGGATACCAGCCTGAATACTGGGGTA NTGVF TTC 813 CGTWDTSL 1637 TGCGGAACATGGGATACCAGCCTGAATATTTGGGTG NIWVF TTC 814 CGTWDTSL 1638 TGCGGAACATGGGATACAAGCCTGAATATTGGGGTG NIGVF TTC 815 CGTWDTSLI 1639 TGCGGAACATGGGATACCAGCCTGATTGCTGTGGTG AVVF TTC 816 CGTWDRSL 1640 TGCGGAACGTGGGATAGGAGCCTGAGTGGTTGGGTG SGWVF TTC 817 CGTWDNRL 1641 TGCGGAACATGGGATAACAGGCTGAGTGGTTGGGTG SGWVF TTC 818 CGTWDKSL 1642 TGCGGAACGTGGGATAAGAGCCTGAGTGCTGTGGTC SAVVF TTC 819 CGTWDKGL 1643 TGCGGAACATGGGATAAAGGCCTGAGTGCTTGGGTG SAWVF TTC 820 CGTWDISLS 1644 TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTG AGVF TTC 821 CGTWDESL 1645 TGCGGAACATGGGATGAGAGCCTGAGTGGTGGCGA SGGEVVF GGTGGTCTTC 822 CGTWDASL 1646 TGCGGAACATGGGATGCCAGCCTGAGTGCCTGGGTG SAWVF TTC 823 CGTWDAGL 1647 TGCGGAACTTGGGATGCCGGCCTGAGTGCTTGGGTG SAWVF TTC 824 CGAWDTSL 1648 TGCGGAGCATGGGATACCAGCCTGAGTGCTTGGGTG SAWVF TTC 825 CGAWDTSL 1649 TGCGGAGCATGGGATACCAGCCTGAGTGCTGTGGTG SAVVF TTC 826 CGAWDTSL 1650 TGCGGAGCATGGGATACCAGCCTGCGTGCTGGGGTT RAGVF TTC 827 CATWDTSV 1651 TGCGCAACATGGGATACCAGCGTGAGTGCTTGGGTG SAWVF TTC 828 CATWDTSL 1652 TGCGCAACATGGGATACCAGCCTGAGTGCGTGGGTG SAWVF TTC 829 CATWDNTL 1653 TGCGCAACATGGGACAACACCCTGAGTGCTGGGGTG SAGVF TTC 830 CAAWDRSL 1654 TGCGCAGCATGGGATAGGAGCCTGAGTGTTTGGGTG SVWVF TTC 831 CYTWHSSL 1655 TGCTACACATGGCATTCCAGTCTGCGTGGTGGGGTG RGGVF TTC 832 CVTWTSSPS 1656 TGCGTAACGTGGACTAGTAGCCCGAGTGCTTGGGTG AWVF TTC 833 CVTWRGGL 1657 TGCGTGACATGGCGTGGTGGCCTTGTGTTGTTC VLF 834 CVTWDTSL 1658 TGCGTAACATGGGATACCAGCCTGACTTCTGTGGTA TSVVL CTC 835 CVTWDTSL 1659 TGCGTAACATGGGATACCAGCCTGAGTGTTTATTGG SVYWVF GTGTTC 836 CVTWDTSL 1660 TGCGTTACATGGGATACCAGCCTGAGTGCCTGGGTG SAWVF TTC 837 CVTWDTDL 1661 TGCGTCACATGGGATACCGACCTCAGCGTTGCGCTC SVALF TTC 838 CVTWDRSL 1662 TGCGTAACATGGGATAGGAGCCTGAGTGGTTGGGTG SGWVF TTC 839 CVTWDRSL 1663 TGCGTAACATGGGATCGCAGCCTGAGAGAGGTGTTA REVLF TTC 840 CVTWDRSL 1664 TGCGTAACATGGGATCGCAGCCTGAGAGCGGTGGTA RAVVF TTC 841 CVTWDRSL 1665 TGCGTAACATGGGACAGGAGCCTCGATGCTGGGGTT DAGVF TTC 842 CVTWDNTL 1666 TGCGTGACATGGGATAACACCCTGAGTGCTGGGGTC SAGVF TTC 843 CVTWDNNL 1667 TGCGTAACATGGGATAACAACCTGTTTGGTGTGGTC FGVVF TTC 844 CVSWDTSL 1668 TGCGTATCATGGGATACCAGCCTGAGTGGTGCGGTA SGAVF TTC 845 CVSWDTSL 1669 TGCGTCTCATGGGATACCAGCCTGAGTGCTGGGGTA SAGVF TTC 846 CTTWFRTPS 1670 TGCACAACATGGTTTAGGACTCCGAGTGATGTGGTC DVVF TTC 847 CTTWFRTA 1671 TGCACAACATGGTTTAGGACTGCGAGTGATGTGGTC SDVVF TTC 848 CTTWDYGL 1672 TGCACAACGTGGGATTACGGTCTGAGTGTCGTCTTC SVVF 849 CTARDTSLS 1673 TGCACAGCAAGGGATACCAGCCTGAGTCCTGGCGGG PGGVF GTCTTC 850 CSTWNTRP 1674 TGCTCAACATGGAATACGAGGCCGAGTGATGTGGTG SDVVF TTC 851 CSTWESSLT 1675 TGTTCAACATGGGAGAGCAGTTTGACTACTGTGGTC TVVF TTC 852 CSTWDTSL 1676 TGCTCAACATGGGATACCAGCCTCACTAATGTGCTA TNVLF TTC 853 CSTWDTSL 1677 TGCTCAACATGGGATACCAGCCTGAGTGGAGTAGTC SGVVF TTC 854 CSTWDHSL 1678 TGCTCAACATGGGATCACAGCCTGAAAGCTGCACTG KAALF TTC 855 CSTWDARL 1679 TGCTCAACCTGGGATGCGAGGCTGAGTGTCCGGGTG SVRVF TTC 856 CSSYTSSST 1680 TGCTCCTCATATACAAGCAGCAGCACTTGGGTGTTC WVF 857 CSSYATRG 1681 TGCAGCTCATACGCAACCCGCGGCCTTCGTGTGTTG LRVLF TTC 858 CSSWDATL 1682 TGTTCATCATGGGACGCCACCCTGAGTGTTCGCATA SVRIF TTC 859 CQVWEGSS 1683 TGTCAGGTGTGGGAGGGTAGTAGTGATCATTGGGTG DHWVF TTC 860 CQTWDNRL 1684 TGCCAAACCTGGGATAACAGACTGAGTGCTGTGGTG SAVVF TTC 861 CQTWDHSL 1685 TGTCAAACGTGGGATCACAGCCTGCATGTTGGGGTG HVGVF TTC 862 CQSYDDILN 1686 TGCCAGTCCTATGACGACATCTTGAATGTTTGGGTCC VWVL TT 863 CNTWDKSL 1687 TGCAATACATGGGATAAGAGTTTGACTTCTGAACTC TSELF TTC 864 CLTWDRSL 1688 TGCTTAACATGGGATCGCAGCCTGAATGTGAGGGTG NVRVF TTC 865 CLTWDHSL 1689 TGCCTAACATGGGACCACAGCCTGACTGCTTATGTC TAYVF TTC 866 CLTRDTSLS 1690 TGCTTAACAAGGGATACCAGTCTGAGTGCCCCTGTG APVF TTC 867 CKTWESGL 1691 TGCAAAACATGGGAAAGTGGCCTTAATTTTGGCCAC NFGHVF GTCTTC 868 CKTWDTSL 1692 TGCAAAACATGGGATACCAGCCTGAGTGCTGTGGTC SAVVF TTC 869 CGVWDVSL 1693 TGCGGAGTCTGGGATGTCAGTCTGGGTGCTGGGGTG GAGVF TTC 870 CGVWDTTP 1694 TGCGGAGTCTGGGATACCACCCCGAGTGCCGTTCTT SAVLF TTC 871 CGVWDTTL 1695 TGCGGAGTCTGGGATACCACCCTGAGTGCCGTTCTT SAVLF TTC 872 CGVWDTSL 1696 TGCGGAGTATGGGATACCAGCCTGGGGGTCTTC GVF 873 CGVWDTNL 1697 TGCGGGGTATGGGATACCAACCTGGGTAAATGGGTT GKWVF TTC 874 CGVWDTGL 1698 TGTGGAGTTTGGGATACTGGCCTGGATGCTGGTTGG DAGWVF GTGTTC 875 CGVWDNV 1699 TGCGGAGTGTGGGATAACGTCCTGGAGGCCTATGTC LEAYVF TTC 876 CGVWDISL 1700 TGCGGAGTCTGGGATATCAGCCTGAGTGCTAATTGG SANWVF GTGTTC 877 CGVWDHSL 1701 TGCGGAGTATGGGATCACAGCCTGGGGATTTGGGCC GIWAF TTC 878 CGVWDDIL 1702 TGCGGAGTTTGGGATGATATTCTGACTGCTGAAGTG TAEVF TTC 879 CGVRDTSL 1703 TGCGGAGTTCGGGATACCAGCCTGGGGGTCTTC GVF 880 CGTYDTSLP 1704 TGCGGAACATACGATACGAGCCTGCCTGCTTGGGTG AWVF TTT 881 CGTYDNLV 1705 TGCGGAACTTACGATAATCTTGTATTTGGTTATGTCT FGYVF TC 882 CGTYDDRL 1706 TGCGGAACATACGATGATAGACTCAGAGAGGTGTTC REVF 883 CGTWVTSL 1707 TGCGGAACGTGGGTTACCAGCCTGAGTGCTGGGGTG SAGVF TTC 884 CGTWVSSL 1708 TGCGGAACATGGGTTAGCAGCCTGACTACTGTAGTA TTVVF TTC 885 CGTWVSSL 1709 TGCGGAACATGGGTTAGCAGCCTGAACGTCTGGGTG NVWVF TTC 886 CGTWVGRF 1710 TGCGGAACATGGGTTGGCAGGTTTTGGGTATTC WVF 887 CGTWSGGP 1711 TGCGGAACATGGTCTGGCGGCCCGAGTGGCCATTGG SGHWLF TTGTTC 888 CGTWSGGL 1712 TGCGGAACATGGTCTGGCGGCCTGAGTGGCCATTGG SGHWLF TTGTTC 889 CGTWQTGR 1713 TGCGGAACGTGGCAGACCGGCCGGGAGGCTGTCCTA EAVLF TTT 890 CGTWQSRL 1714 TGCGGAACGTGGCAGAGCAGGCTGAGGTGGGTGTTC RWVF 891 CGTWQSRL 1715 TGCGGAACGTGGCAGAGCAGGCTGGGGTGGGTGTTC GWVF 892 CGTWPRSL 1716 TGCGGAACATGGCCTAGGAGCCTGAGTGCTGTTTGG SAVWVF GTGTTC 893 CGTWNNYL 1717 TGCGGAACATGGAATAACTACCTGAGTGCTGGCGAT SAGDVVF GTGGTTTTC 894 CGTWLGSQ 1718 TGCGGAACATGGCTTGGCAGCCAGAGTCCTTATTGG SPYWVF GTCTTC 895 CGTWHTGL 1719 TGCGGAACATGGCATACCGGCCTGAGTGCTTATGTC SAYVF TTC 896 CGTWHSTL 1720 TGCGGAACATGGCATAGTACCCTGAGTGCTGGCCAT SAGHWVF TGGGTGTTC 897 CGTWHSSL 1721 TGCGGAACATGGCATAGTAGCCTGAGTACTTGGGTG STWVF TTC 898 CGTWHSSL 1722 TGCGGAACATGGCATAGCAGCCTGAGTGCCTATGTC SAYVF TTC 899 CGTWHSSL 1723 TGCGGAACATGGCATAGCAGCCTGAGTGCTGTGGTA SAVVF TTC 900 CGTWHSGL 1724 TGCGGAACGTGGCATTCCGGCCTGAGTGGGTGGGTT SGWVF TTC 901 CGTWHNTL 1725 TGCGGAACATGGCATAACACCCTGCGTAATGTGATA RNVIF TTC 902 CGTWHASL 1726 TGCGGAACATGGCATGCCAGCCTGACTGCTGTGTTC TAVF 903 CGTWGWY 1727 TGCGGGACATGGGGATGGTATGGCAGCCAGAGAGG GSQRGVVF CGTCGTCTTC 904 CGTWGWY 1728 TGCGGGACATGGGGATGGTATGGCGGCCAGAGAGG GGQRGVVF CGTCGTCTTC 905 CGTWGTSL 1729 TGCGGAACCTGGGGAACCAGCCTGAGTGCTTGGGTG SAWVF TTC 906 CGTWGSSL 1730 TGCGGAACCTGGGGTAGCAGCCTGACTACTGGCCTG TTGLF TTC 907 CGTWGSSL 1731 TGCGGAACATGGGGTAGCAGCCTGACTGCCTATGTC TAYVF TTC 908 CGTWGSSL 1732 TGCGGAACATGGGGTAGCAGCCTGAGTGTTGTGTTC SVVF 909 CGTWGSSL 1733 TGCGGAACATGGGGTAGCAGCCTGAGTGGTGGGGT SGGVF GTTC 910 CGTWGSSL 1734 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATTGG SAYWVF GTGTTC 911 CGTWGSSL 1735 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTG SAYVVF GTGTTC 912 CGTWGSSL 1736 TGCGGAACATGGGGTAGCAGCCTGAGTGCTTATGTC SAYVF TTC 913 CGTWGSSL 1737 TGCGGAACGTGGGGTAGTAGCCTGAGTGCTGTGGTG SAVVF TTC 914 CGTWGSSL 1738 TGCGGAACATGGGGTAGCAGCCTGAGTGCTCCTTAT SAPYVF GTCTTC 915 CGTWGSSL 1739 TGCGGAACATGGGGTAGCAGCCTGAGTGCCCCGGTG SAPVF TTC 916 CGTWGSSL 1740 TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGTG SAGVF TTC 917 CGTWGSSL 1741 TGCGGAACTTGGGGTAGCAGCCTGAGTGCTGGACTG SAGLF TTC 918 CGTWGSSL 1742 TGCGGAACATGGGGTAGCAGCCTGAGTGCTGGGGC SAGALF ACTCTTC 919 CGTWGSSL 1743 TGCGGAACATGGGGCAGTAGCCTGCGTGCTTGGGTG RAWVF TTC 920 CGTWFTSL 1744 TGCGGAACCTGGTTTACTAGTCTGGCTAGTGGGGTT ASGVF TTC 921 CGTWETSL 1745 TGCGGAACTTGGGAGACCAGTCTGAGTGTCGTGGTC SVVVI ATC 922 CGTWETSL 1746 TGCGGAACATGGGAGACCAGCCTGAGTGGTGTCTTC SGVF 923 CGTWETSL 1747 TGCGGAACATGGGAAACCAGCCTGAGTGATTGGGTA SDWVF TTC 924 CGTWETSL 1748 TGCGGAACATGGGAGACCAGCCTGAGTGCTGGGGT SAGVF ATTC 925 CGTWETSL 1749 TGCGGAACATGGGAAACCAGCCTTAATTATGTGGCC NYVAF TTC 926 CGTWETSL 1750 TGCGGAACATGGGAGACCAGCCTGAATACTTGGTTG NTWLL CTC 927 CGTWETSE 1751 TGCGGAACATGGGAGACCAGCGAGAGTGGTAATTA SGNYIF CATCTTC 928 CGTWETRL 1752 TGCGGAACATGGGAAACCAGACTGGGTACTTGGGTG GTWVI ATC 929 CGTWETQL 1753 TGCGGAACATGGGAGACCCAGTTATATTGGGTGTTC YWVF 930 CGTWETGL 1754 TGCGGAACATGGGAGACTGGCCTAAGTGCTGGAGA SAGEVF GGTGTTC 931 CGTWESTL 1755 TGCGGAACTTGGGAAAGCACCCTGAGTGTTTTCCTA SVFLF TTC 932 CGTWESSL 1756 TGCGGGACATGGGAAAGTAGCCTGACTGTTGTGGTC TVVVF TTC 933 CGTWESSL 1757 TGCGGAACATGGGAAAGTAGCCTGACTGGAGTGGT TGVVF ATTC 934 CGTWESSL 1758 TGCGGAACATGGGAAAGCAGCCTGACTGGTTTTGTC TGFVF TTC 935 CGTWESSL 1759 TGTGGAACATGGGAGAGCAGCCTGAGTGTTGGGGTG SVGVF TTC 936 CGTWESSL 1760 TGCGGAACCTGGGAAAGTAGCCTCAGTGAATGGGTG SEWVF TTC 937 CGTWESSL 1761 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGTATTC SAVF 938 CGTWESSL 1762 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGTTAT SAGYIF ATCTTC 939 CGTWESSL 1763 TGCGGAACATGGGAGAGCAGCCTGAGTGCTGGAGT SAGVF GTTC 940 CGTWESSL 1764 TGCGGAACATGGGAAAGCAGCCTGAGCGCTGGCCC SAGPVF GGTGTTC 941 CGTWESSL 1765 TGCGGAACATGGGAAAGCAGCCTGAGTGCTGGAGG SAGGQVF CCAGGTGTTC 942 CGTWESSL 1766 TGCGGAACATGGGAGAGCAGCCTGAGTGCCTTCGGC SAFGGYVF GGTTATGTCTTC 943 CGTWESSL 1767 TGCGGAACATGGGAAAGCAGCCTGAGGGTTTGGGT RVWVF GTTC 944 CGTWESSL 1768 TGCGGAACATGGGAAAGCAGCCTCTTTACTGGGCCT FTGPWVF TGGGTGTTC 945 CGTWESLS 1769 TGCGGAACATGGGAGAGCCTGAGTGCCACCTATGTC ATYVF TTC 946 CGTWESGL 1770 TGCGGAACATGGGAGAGCGGCCTGAGTGCTGGTGTC SAGVF TTC 947 CGTWESDF 1771 TGCGGAACATGGGAAAGCGACTTTTGGGTGTTT WVF 948 CGTWENRL 1772 TGCGGTACATGGGAAAACAGACTGAGTGCTGTGGTC SAVVF TTC 949 CGTWENRL 1773 TGCGGAACATGGGAAAACAGACTGAGTGCCGGGGT SAGVF ATTC 950 CGTWEISLT 1774 TGCGGAACATGGGAAATCAGCCTGACTACTTCTGTG TSVVF GTATTC 951 CGTWEISLS 1775 TGCGGAACATGGGAAATCAGCCTGAGTACTTCTGTG TSVVF GTATTC 952 CGTWEGSL 1776 TGCGGAACATGGGAAGGCAGCCTCAGTGTTGTTTTC SVVF 953 CGTWEGSL 1777 TGCGGAACATGGGAAGGCAGCCTGAGGGTGTTC RVF 954 CGTWEGSL 1778 TGCGGAACATGGGAGGGCAGCCTGAGGCACGTGTTC RHVF 955 CGTWDYSP 1779 TGCGGAACATGGGATTACAGCCCTGTACGTGCTGGG VRAGVF GTGTTC 956 CGTWDYSL 1780 TGCGGAACGTGGGATTACAGCCTGAGTGTTTATCTC SVYLF TTC 957 CGTWDYSL 1781 TGCGGAACATGGGATTACAGCCTGAGTTCTGGCGTG SSGVVF GTATTC 958 CGTWDYSL 1782 TGCGGAACATGGGATTACAGCCTGAGTGCCTGGGTG SAWVF TTC 959 CGTWDYSL 1783 TGCGGAACATGGGATTACAGTCTGAGTGCTGAGGTG SAEVF TTC 960 CGTWDYSL 1784 TGCGGAACATGGGATTACAGCCTGCGTCGTGCGATA RRAIF TTC 961 CGTWDWSL 1785 TGCGGAACATGGGATTGGAGCCTCATTCTTCAATTG ILQLF TTC 962 CGTWDVTL 1786 TGCGGAACATGGGATGTCACCTTGCATACTGGGGTG HTGVF TTC 963 CGTWDVTL 1787 TGCGGAACATGGGATGTCACCTTGCATATTGGGGTG HIGVF TTC 964 CGTWDVTL 1788 TGCGGAACATGGGATGTCACCTTGCATGCTGGGGTG HAGVF TTC 965 CGTWDVSL 1789 TGCGGAACATGGGATGTCAGTTTGTATAGTGGCGGG YSGGVF GTCTTC 966 CGTWDVSL 1790 TGTGGAACATGGGATGTCAGCCTGACTTCTTTCGTCT TSFVF TC 967 CGTWDVSL 1791 TGCGGAACATGGGATGTCAGCCTGAGTGTTGGGGTG SVGVL CTC 968 CGTWDVSL 1792 TGCGGAACGTGGGATGTCAGCCTGAGTGCTGGCGAT SAGDVVF GTAGTTTTC 969 CGTWDVSL 1793 TGCGGAACATGGGATGTCAGCCTGAATGTCGTGGTT NVVVF TTC 970 CGTWDVSL 1794 TGCGGAACATGGGATGTCAGCCTGAATACTCAGGTG NTQVF TTC 971 CGTWDVSL 1795 TGCGGCACATGGGATGTGAGCCTGGGTGCGCTGTTC GALF 972 CGTWDVNL 1796 TGCGGAACGTGGGACGTTAATCTGAAAACTGTCGTT KTVVF TTC 973 CGTWDVIL 1797 TGCGGAACATGGGATGTCATCCTGAGTGCTGAGGTA SAEVF TTC 974 CGTWDTTV 1798 TGCGGAACATGGGATACCACCGTGAGTGCTGTGGTT SAVVF TTC 975 CGTWDTTL 1799 TGCGGAACATGGGATACCACCCTGACTGCCTGGGTG TAWVF TTC 976 CGTWDTTL 1800 TGCGGAACATGGGACACCACCTTGAGTGTTTTCCTA SVFLF TTC 977 CGTWDTSV 1801 TGCGGGACTTGGGATACCAGTGTGAGTGCTGGGGTG SAGVF TTC 978 CGTWDTSV 1802 TGCGGAACATGGGATACCAGTGTGATTTCTTGGGTT ISWVF TTC 979 CGTWDTSR 1803 TGCGGAACATGGGATACCAGTCGGAGTTCTCTCTAT SSLYVVF GTGGTCTTC 980 CGTWDTSR 1804 TGCGGAACATGGGATACCAGCCGGAGTGCTTGGGTA SAWVF TTC 981 CGTWDTSR 1805 TGCGGAACATGGGATACCAGCCGGAATCCTGGAGG NPGGIF AATTTTC 982 CGTWDTSR 1806 TGCGGAACATGGGACACCAGTCGGGGTCATGTTTTC GHVF 983 CGTWDTSP 1807 TGCGGAACATGGGATACCAGCCCGAGTACTGGCCAG STGQVLF GTGCTTTTC 984 CGTWDTSP 1808 TGCGGAACATGGGATACCAGCCCGAGTGCCTGGGTG SAWVF TTC 985 CGTWDTSL 1809 TGCGGAACATGGGATACTAGCCTGACCTGGGTGTTC TWVF 986 CGTWDTSL 1810 TGCGGAACATGGGATACCAGCCTGACGTGGTTCGCA TWFAVF GTGTTC 987 CGTWDTSL 1811 TGCGGAACATGGGATACCAGCCTGACTGTTGTGGTA TVVVF TTC 988 CGTWDTSL 1812 TGCGGAACATGGGATACCAGCCTGACTACTTCTTGG TTSWVF GTGTTC 989 CGTWDTSL 1813 TGCGGAACATGGGATACCAGCCTGACCACTGGTCCT TTGPFWVF TTTTGGGTGTTC 990 CGTWDTSL 1814 TGCGGAACATGGGATACCAGCCTGACTCCTTTTTAT TPFYVF GTCTTC 991 CGTWDTSL 1815 TGCGGAACATGGGATACCAGCCTGACTGCTTATGTC TAYVF TTC 992 CGTWDTSL 1816 TGCGGAACATGGGATACCAGCCTGACTGCTTGGGTG TAWVF TTC 993 CGTWDTSL 1817 TGCGGAACATGGGATACCAGCCTGACTGCGTGGGGG TAWGVF GTGTTC 994 CGTWDTSL 1818 TGCGGCACATGGGATACCAGCCTGACTGCGGTGGTT TAVVL CTC 995 CGTWDTSL 1819 TGCGGAACCTGGGATACCAGCCTGACTGCTCGGGTT TARVF TTC 996 CGTWDTSL 1820 TGCGGAACATGGGATACCAGCCTGACTGCGATTGTC TAIVF TTC 997 CGTWDTSL 1821 TGCGGAACATGGGATACCAGCCTGACTGCTGGTGTC TAGVF TTC 998 CGTWDTSL 1822 TGCGGAACATGGGATACCAGCCTGAGTGTTTATGTC SVYVF TTC 999 CGTWDTSL 1823 TGCGGAACATGGGATACCAGCCTGAGTGTGGTGTTC SVVF 1000 CGTWDTSL 1824 TGCGGGACATGGGATACCAGCCTGAGTGTTGGGGAA SVGEF TTC 1001 CGTWDTSL 1825 TGCGGAACATGGGATACCAGCCTGAGTACTTGGGTG STWVF TTC 1002 CGTWDTSL 1826 TGCGGAACATGGGATACCAGCCTGAGTACTGTGGTA STVVF TTC 1003 CGTWDTSL 1827 TGCGGAACATGGGATACCAGCCTGAGTACTGGCCAG STGQVLF GTGCTTTTC 1004 CGTWDTSL 1828 TGCGGCACATGGGATACCAGCCTGAGCACTGGTCCT STGPLWVF CTTTGGGTGTTC 1005 CGTWDTSL 1829 TGCGGAACTTGGGATACCAGCCTGAGTTCTTATGTC SSYVF TTC 1006 CGTWDTSL 1830 TGCGGAACATGGGATACCAGCCTGAGTTCTGTGGTC SSVVF TTC 1007 CGTWDTSL 1831 TGCGGAACATGGGATACCAGCCTGAGTTCTAGATAC SSRYIF ATATTC 1008 CGTWDTSL 1832 TGCGGAACATGGGATACCAGCCTGAGTTCTAGATTC SSRFIF ATATTC 1009 CGTWDTSL 1833 TGCGGAACATGGGATACCAGCCTGAGTTCTGGGTGG SSGWVF GTGTTC 1010 CGTWDTSL 1834 TGCGGAACATGGGATACCAGCCTGAGTCGGTATGTG SRYVF TTC 1011 CGTWDTSL 1835 TGCGGAACTTGGGATACCAGTCTGAGTCAATGGCTG SQWLF TTC 1012 CGTWDTSL 1836 TGCGGAACATGGGATACCAGCCTGAGTCCTGGCCTT SPGLWVF TGGGTGTTC 1013 CGTWDTSL 1837 TGCGGAACATGGGATACCAGCCTGAGTAATTATGTC SNYVF TTC 1014 CGTWDTSL 1838 TGCGGAACATGGGATACCAGCCTAAGTATTTGGGTG SIWVF TTC 1015 CGTWDTSL 1839 TGCGGCACATGGGATACCAGCCTGAGCATTGGTCCT SIGPFWVF TTTTGGGTGTTC 1016 CGTWDTSL 1840 TGCGGAACATGGGATACCAGCCTGAGTGGTTGGGTG SGWVF TTC 1017 CGTWDTSL 1841 TGCGGAACATGGGATACCAGCCTGAGTGGTACAGTG SGTVF TTC 1018 CGTWDTSL 1842 TGCGGAACATGGGATACTAGTCTGAGTGGTGGCCAG SGGQVF GTGTTC 1019 CGTWDTSL 1843 TGCGGAACATGGGATACCAGCCTGAGTGGTGGGATA SGGIF TTC 1020 CGTWDTSL 1844 TGCGGAACATGGGATACCAGCCTGAGTGGTGAGGAT SGEDVVI GTGGTAATC 1021 CGTWDTSL 1845 TGCGGAACATGGGATACCAGCCTGAGTTTCCTTTAT SFLYAF GCTTTC 1022 CGTWDTSL 1846 TGCGGAACATGGGATACCAGCCTGAGTGAGGTCGTA SEVVF TTC 1023 CGTWDTSL 1847 TGCGGAACATGGGATACCAGCCTGAGTGAAGTGTTC SEVF 1024 CGTWDTSL 1848 TGCGGAACATGGGATACTAGCCTGAGTGAAAATTGG SENWVF GTGTTC 1025 CGTWDTSL 1849 TGCGGAACATGGGATACCAGCCTGAGTGCCTACATA SAYIF TTC 1026 CGTWDTSL 1850 TGCGGAACATGGGATACCAGCCTGAGTGCTGTGGTA SAVVL CTC 1027 CGTWDTSL 1851 TGCGGAACATGGGATACCAGCCTGAGTGCTGTTTTC SAVF 1028 CGTWDTSL 1852 TGCGGAACATGGGATACCAGCCTGAGTGCCCGGGTG SARVF TTC 1029 CGTWDTSL 1853 TGCGGCACATGGGATACCAGCCTGAGTGCCCGCCAG SARQVF GTATTC 1030 CGTWDTSL 1854 TGCGGAACATGGGATACCAGCCTGAGTGCTTTGGTT SALVF TTC 1031 CGTWDTSL 1855 TGCGGAACATGGGATACCAGCCTGAGTGCTAAGGTG SAKVF TTC 1032 CGTWDTSL 1856 TGCGGAACATGGGATACCAGCCTGAGTGCGAAAATC SAKIF TTC 1033 CGTWDTSL 1857 TGCGGAACATGGGATACCAGCCTGAGTGCCAAGGC SAKAVF GGTATTC 1034 CGTWDTSL 1858 TGCGGAACATGGGATACCAGCCTGAGTGCCCATGCT SAHAVF GTGTTC 1035 CGTWDTSL 1859 TGCGGAACATGGGATACCAGCCTGAGTGCTGGCTAT SAGYVF GTCTTC 1036 CGTWDTSL 1860 TGCGGAACATGGGACACCAGTCTGAGTGCTGGCCGC SAGRWVF TGGGTGTTC 1037 CGTWDTSL 1861 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGATA SAGIF TTC 1038 CGTWDTSL 1862 TGCGGAACATGGGATACCAGCCTGAGTGCTGGTGGG SAGGFRVF TTCCGGGTCTTC 1039 CGTWDTSL 1863 TGCGGAACATGGGATACCAGCCTGAGTGCTGGGGCA SAGAF TTC 1040 CGTWDTSL 1864 TGCGGAACATGGGATACCAGTCTGAGTGCTGATTGG SADWFF TTTTTC 1041 CGTWDTSL 1865 TGCGGAACATGGGATACCAGCCTGAGTGCTGATGAA SADEYVF TATGTCTTC 1042 CGTWDTSL 1866 TGCGGCACATGGGATACCAGCCTGAGTGCGGCTTGG SAAWVF GTGTTC 1043 CGTWDTSL 1867 TGCGGAACATGGGATACCAGCCTGAGTGCTGCGCTA SAALF TTC 1044 CGTWDTSL 1868 TGCGGAACATGGGATACCAGCCTGAGTGCTGCGGGG SAAGVF GTTTTC 1045 CGTWDTSL 1869 TGCGGAACATGGGATACCAGCCTGAGAGTTGTGGTT RVVVF TTC 1046 CGTWDTSL 1870 TGCGGAACATGGGATACCAGCCTGAGAACCTGGGTA RTWVF TTC 1047 CGTWDTSL 1871 TGCGGAACGTGGGATACCAGCCTGAGGGGTGCAGT RGAVF GTTC 1048 CGTWDTSL 1872 TGCGGAACATGGGATACCAGCCTGCGTGCTGTGGTA RAVVF TTC 1049 CGTWDTSL 1873 TGCGGAACATGGGATACAAGCCTGAATGTAGTTTAT NVVYVF GTCTTC 1050 CGTWDTSL 1874 TGCGGAACATGGGATACCAGCCTCAACACCTACCTG NTYLF TTC 1051 CGTWDTSL 1875 TGCGGAACATGGGATACTAGCCTGAACTTCGCTTGG NFAWLF CTGTTC 1052 CGTWDTSL 1876 TGCGGCACATGGGATACCAGCCTTCTTGTGTGGCTTT LVWLF TC 1053 CGTWDTSL 1877 TGCGGAACATGGGATACCAGTCTGAAGACGTGGGTG KTWVF TTC 1054 CGTWDTSLI 1878 TGCGGAACATGGGATACCAGTCTGATTGTCTGGGTG VWVF TTC 1055 CGTWDTSLI 1879 TGCGGAACATGGGATACCAGCCTAATTACTGGGGTG TGVF TTC 1056 CGTWDTSLI 1880 TGCGGAACATGGGATACCAGCCTGATTAGCGTGGTA SVVF TTC 1057 CGTWDTSLI 1881 TGCGGAACATGGGATACCAGCCTGATTGCTTATGTC AYVF TTC 1058 CGTWDTSL 1882 TGCGGAACATGGGATACCAGCCTGCACACTGAGTTG HTELF TTC 1059 CGTWDTSL 1883 TGCGGAACTTGGGATACCAGCCTGGGTTCTTATGTC GSYVF TTC 1060 CGTWDTSL 1884 TGCGGAACATGGGATACCAGCCTGGGTTCTCTTTGG GSLWVF GTGTTC 1061 CGTWDTSL 1885 TGCGGTACATGGGATACCAGCCTGGGTTCTGGGGTA GSGVF TTC 1062 CGTWDTSL 1886 TGCGGAACTTGGGATACCAGTCTGGGTGGTAGAGGG GGRGVF GTCTTC 1063 CGTWDTSL 1887 TGCGGAACATGGGATACCAGCCTGGGTGCTTGGGTG GAWVF TTC 1064 CGTWDTSL 1888 TGCGGAACATGGGATACCAGCCTGGGTGCCGTGGTA GAVVF TTC 1065 CGTWDTSL 1889 TGCGGAACATGGGATACCAGCCTGGGTGCTGGGGTA GAGVF TTC 1066 CGTWDTSL 1890 TGCGGAACATGGGATACCAGCCTGGGTGCTGGCCTA GAGLF TTC 1067 CGTWDTSL 1891 TGCGGAACATGGGATACCAGTCTGGATGCTGTGGTT DAVVF TTC 1068 CGTWDTSL 1892 TGCGGGACTTGGGATACCAGCCTGGATGCTGTGCTG DAVLF TTC 1069 CGTWDTSL 1893 TGCGGAACATGGGATACCAGCCTGGCTTGGGTGTTC AWVF 1070 CGTWDTSL 1894 TGCGGAACATGGGATACCAGCCTGGCGACTGGACTG ATGLF TTC 1071 CGTWDTSL 1895 TGCGGGACATGGGATACCAGCCTGGCCCCTGTAGTC APVVF TTC 1072 CGTWDTRL 1896 TGCGGAACATGGGACACCCGCCTGACTATTGTGATC TIVIF TTC 1073 CGTWDTRL 1897 TGTGGAACATGGGACACCAGGCTGAGTGTTTGGCTG SVWLF TTC 1074 CGTWDTRL 1898 TGCGGAACGTGGGACACCAGACTGAGTGTTGGGGTT SVGVF TTC 1075 CGTWDTRL 1899 TGCGGCACATGGGATACCAGACTGAGTACTGTAATT STVIF TTC 1076 CGTWDTRL 1900 TGCGGAACATGGGATACCCGCCTGAGTTCTGTGGTC SSVVF TTC 1077 CGTWDTRL 1901 TGCGGAACATGGGATACCCGCCTGAGTATTGTGGTT SIVVF TTC 1078 CGTWDTRL 1902 TGCGGAACATGGGATACCAGACTGAGTGCCTATGTG SAYVVF GTATTC 1079 CGTWDTRL 1903 TGCGGAACCTGGGACACCCGCCTGAGTGCGTGGGTG SAWVF TTC 1080 CGTWDTRL 1904 TGCGGAACATGGGATACCAGACTGAGTGCTGTGGTG SAVVF TTC 1081 CGTWDTRL 1905 TGCGGAACATGGGATACCCGCCTGAGTGCTGGGTTG SAGLF TTC 1082 CGTWDTRL 1906 TGCGGAACATGGGATACCAGACTGAGTGCTGGTGGG SAGGVF GTGTTC 1083 CGTWDTRL 1907 TGCGGAACATGGGATACCAGATTGAATGTGTGGCTA NVWLF TTC 1084 CGTWDTNR 1908 TGCGGAACATGGGATACCAACCGGGAAGTTGTGCTC EVVLL CTC 1085 CGTWDTNL 1909 TGCGGAACATGGGATACCAACCTGCGTGCCCATGTC RAHVF TTC 1086 CGTWDTNL 1910 TGCGGAACATGGGATACTAATCTGCCCGCTGTAGTG PAVVF TTC 1087 CGTWDTNL 1911 TGCGGAACATGGGACACCAATTTGGGTGGGGTGTTC GGVF 1088 CGTWDTIV 1912 TGCGGAACATGGGATACCATCGTGAGTATTGGGGTG SIGVF TTC 1089 CGTWDTILS 1913 TGCGGAACATGGGATACCATCCTGAGTGCGGTGGTG AVVF TTC 1090 CGTWDTILS 1914 TGCGGCACATGGGATACCATCCTGAGTGCTGAGGTG AEVF TTC 1091 CGTWDTHL 1915 TGCGGAACATGGGATACCCACCTGGGTGTGGTTTTC GVVF 1092 CGTWDTGP 1916 TGCGGAACATGGGATACCGGCCCGAGCCCTCATTGG SPHWLF CTGTTC 1093 CGTWDTGL 1917 TGCGGAACATGGGATACCGGCCTGACTTTTGGAGGC TFGGVF GTGTTC 1094 CGTWDTGL 1918 TGCGGAACATGGGATACCGGCCTGACTGCTTTTGTC TAFVF TTC 1095 CGTWDTGL 1919 TGCGGAACATGGGATACCGGCCTGAGTGTTTGGGTG SVWVF TTC 1096 CGTWDTGL 1920 TGCGGAACATGGGATACCGGCCTGAGTACTGGGATT STGIF TTC 1097 CGTWDTGL 1921 TGCGGAACATGGGATACCGGCCTGAGTTCCCTGCTC SSLLF TTC 1098 CGTWDTGL 1922 TGCGGAACGTGGGACACCGGCCTGAGTATTGTGGTG SIVVF TTC 1099 CGTWDTGL 1923 TGCGGAACGTGGGACACCGGCCTGAGTTTTGTGGTG SFVVF TTC 1100 CGTWDTGL 1924 TGCGGAACATGGGATACCGGCCTGAGTGCTTGGGTG SAWVF TTC 1101 CGTWDTGL 1925 TGCGGAACATGGGATACCGGCCTGAGTGCTGGTGTG SAGVVF GTATTC 1102 CGTWDTGL 1926 TGCGGAACATGGGATACCGGTCTGAGGGGTTGGATT RGWIF TTC 1103 CGTWDTEL 1927 TGCGGAACATGGGATACCGAGCTAAGTGCGGGGGT SAGVF CTTC 1104 CGTWDTAL 1928 TGCGGAACGTGGGATACCGCCCTGACTGCTGGGGTG TAGVF TTC 1105 CGTWDTAL 1929 TGCGGAACATGGGATACTGCCCTGAGTCTTGTGGTC SLVVF TTC 1106 CGTWDTAL 1930 TGCGGAACATGGGATACCGCCCTGAGTGCCTGGCTG SAWLF TTC 1107 CGTWDTAL 1931 TGCGGCACATGGGATACCGCCCTGAGTGCTGGGGTG SAGVF TTC 1108 CGTWDTAL 1932 TGCGGAACATGGGATACCGCCCTGCGTGGCGTGCTG RGVLF TTC 1109 CGTWDTAL 1933 TGCGGAACATGGGATACCGCCCTGAAAGAATGGCTG KEWLF TTC 1110 CGTWDRTL 1934 TGCGGAACATGGGATAGGACCCTGACTGCTGGCGAT TAGDVLF GTGCTCTTC 1111 CGTWDRSV 1935 TGCGGAACATGGGATAGAAGCGTGACTTATGTCTTC TYVF 1112 CGTWDRSR 1936 TGCGGAACATGGGATCGCAGCCGAAATGAATGGGT NEWVF GTTC 1113 CGTWDRSL 1937 TGCGGAACATGGGATCGCAGTCTGACTGTTTGGGTC TVWVF TTC 1114 CGTWDRSL 1938 TGCGGAACATGGGATCGCAGCCTGACTCCTGGGTGG TPGWLF TTGTTC 1115 CGTWDRSL 1939 TGCGGAACATGGGATAGAAGCCTGACTGCTTGGGTG TAWVF TTC 1116 CGTWDRSL 1940 TGCGGAACATGGGACCGCAGCCTGAGTGTTGTGGTA SVVVF TTC 1117 CGTWDRSL 1941 TGCGGCACATGGGATCGCAGCCTGAGTGTAGTCTTC SVVF 1118 CGTWDRSL 1942 TGCGGAACATGGGATAGGAGCCTGAGTGTTCAATTG SVQLF TTC 1119 CGTWDRSL 1943 TGCGGAACATGGGATCGCAGCCTCAGTGTTCTTTGG SVLWVF GTGTTC 1120 CGTWDRSL 1944 TGCGGAACATGGGATCGCAGCCTGAGTGTTGGATTA SVGLF TTC 1121 CGTWDRSL 1945 TGCGGAACATGGGATCGCAGCCTGAGTACTTGGGTG STWVF TTC 1122 CGTWDRSL 1946 TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGG STHWVL GTGCTC 1123 CGTWDRSL 1947 TGCGGAACATGGGATAGAAGCCTGAGTACTCATTGG STHWVF GTGTTC 1124 CGTWDRSL 1948 TGCGGAACCTGGGATCGAAGCCTGAGTTCTGCGGTG SSAVF TTC 1125 CGTWDRSL 1949 TGCGGAACATGGGACAGAAGCCTGAGTCCCTCTTAT SPSYVF GTCTTC 1126 CGTWDRSL 1950 TGCGGAACATGGGATAGGAGCCTGAGTGGTGAGGT SGEVF GTTC 1127 CGTWDRSL 1951 TGCGGAACATGGGATAGGAGCCTGAGTGGTGCGGT SGAVF GTTC 1128 CGTWDRSL 1952 TGCGGAACATGGGATCGCAGCCTGAGTGCTGTGGCA SAVAF TTC 1129 CGTWDRSL 1953 TGCGGAACATGGGATAGGAGCCTGAGTGCCGGGGG SAGGEF GGAATTC 1130 CGTWDRSL 1954 TGCGGAACATGGGATCGCAGCCTGAGTGCTTTTTGG SAFWVF GTGTTC 1131 CGTWDRSL 1955 TGCGGAACATGGGATAGGAGCCTGAGTGCTGCGGTG SAAVF TTC 1132 CGTWDRSL 1956 TGCGGAACATGGGATAGGAGCCTGAGTGCTGCACTC SAALF TTC 1133 CGTWDRSL 1957 TGCGGAACATGGGATCGCAGCCTGAGAGTGTTC RVF 1134 CGTWDRSL 1958 TGCGGTACATGGGACAGAAGCCTTAATTGGGTGTTC NWVF 1135 CGTWDRSL 1959 TGCGGAACATGGGATCGCAGCCTGAATGTTTATGTC NVYVF TTC 1136 CGTWDRSL 1960 TGCGGAACATGGGATAGGAGCCTGAATGTTGGGGTG NVGVF TTC 1137 CGTWDRSL 1961 TGCGGAACATGGGATCGGAGCCTGCATGTGGTCTTC HVVF 1138 CGTWDRSL 1962 TGTGGAACATGGGATCGCAGCCTGGGTGGTTGGGTG GGWVF TTC 1139 CGTWDRSL 1963 TGCGGAACATGGGATCGCAGCCTGGGTGCTTTTTGG GAFWVF GTGTTC 1140 CGTWDRSL 1964 TGCGGAACATGGGATAGAAGCCTGTTTTGGGTGTTC FWVF 1141 CGTWDRSL 1965 TGCGGAACGTGGGATCGCAGCCTGGCTGCTGGGGTG AAGVF TTC 1142 CGTWDRRL 1966 TGCGGAACATGGGATAGGAGGTTGAGTGGTGTCGTA SGVVF TTC 1143 CGTWDRRL 1967 TGCGGAACGTGGGATCGCCGCCTAAGTGATGTGGTA SDVVF TTC 1144 CGTWDRRL 1968 TGCGGAACATGGGATAGGAGGCTGAGTGCTGTGGTA SAVVF TTC 1145 CGTWDRRL 1969 TGCGGAACATGGGATAGACGCCTGAATGTTGCGTTC NVAFF TTC 1146 CGTWDRRL 1970 TGTGGAACATGGGATAGGAGGCTGCTTGCTGTTTTC LAVF 1147 CGTWDRNL 1971 TGCGGAACTTGGGATAGGAACCTGCGCGCCGTGGTC RAVVF TTC 1148 CGTWDRLS 1972 TGCGGAACATGGGATAGGCTGAGTGCTGGGGTGTTC AGVF 1149 CGTWDRGP 1973 TGCGGAACATGGGATAGAGGCCCGAATACTGGGGT NTGVF ATTC 1150 CGTWDRGL 1974 TGCGGAACATGGGATAGAGGCCTGAATACTGTTTAC NTVYVF GTCTTC 1151 CGTWDNY 1975 TGCGGAACATGGGATAACTATGTGAGTGCCCCTTGG VSAPWVF GTGTTC 1152 CGTWDNYL 1976 TGCGGAACATGGGATAACTACCTGAGTGCTGGCGAT SAGDVVF GTGGTTTTC 1153 CGTWDNYL 1977 TGCGGAACATGGGATAACTACCTGAGAGCTGGGGTC RAGVF TTC 1154 CGTWDNYL 1978 TGCGGAACATGGGACAATTATCTGGGTGCCGTGGTT GAVVF TTC 1155 CGTWDNYL 1979 TGCGGAACATGGGATAACTACCTGGGTGCGGGGGTG GAGVF TTC 1156 CGTWDNTV 1980 TGCGGAACATGGGATAACACCGTGAGTGCCCCTTGG SAPWVF GTTTTC 1157 CGTWDNTL 1981 TGCGGAACATGGGATAACACCCTGAGTCTTTGGGTG SLWVF TTC 1158 CGTWDNTL 1982 TGCGGAACATGGGATAACACCCTGAGTGCTGGGGTC SAGVF TTC 1159 CGTWDNTL 1983 TGCGGAACATGGGACAACACTCTGCTTACTGTGTTA LTVLF TTC 1160 CGTWDNRL 1984 TGCGGAACATGGGATAACAGACTGAGTAGTGTGATT SSVIF TTC 1161 CGTWDNRL 1985 TGCGGAACATGGGATAACAGGTTGAGTGCTGTGGTC SAVVF TTC 1162 CGTWDNRL 1986 TGCGGAACATGGGATAACAGGCTGAGTGCTGGTGG SAGGIF GATATTC 1163 CGTWDNRL 1987 TGCGGAACATGGGATAACAGACTGAGTGCTGAGGT SAEVF GTTC 1164 CGTWDNRL 1988 TGTGGAACATGGGATAACAGACTGCGTGTTGGGGTT RVGVL CTC 1165 CGTWDNRL 1989 TGCGGAACATGGGATAATCGCCTGCTTGAGAATGTC LENVF TTC 1166 CGTWDNNL 1990 TGCGGAACATGGGATAACAACCTGCGTGCTGTCTTC RAVF 1167 CGTWDNNL 1991 TGCGGAACTTGGGATAATAACCTGCGTGCTGGAGTG RAGVF TTC 1168 CGTWDNNL 1992 TGCGGAACATGGGACAACAATTTGGGCGGTGGCCG GGGRVF GGTGTTC 1169 CGTWDNNL 1993 TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTC GAGVL CTC 1170 CGTWDNNL 1994 TGCGGAACATGGGATAACAACCTGGGTGCTGGCGTC GAGVF TTC 1171 CGTWDNIL 1995 TGCGGAACTTGGGATAACATCCTGAGCGCTGCGGTG SAAVF TTC 1172 CGTWDNIL 1996 TGCGGAACCTGGGATAACATCTTGGATGCAGGGGTT DAGVF TTC 1173 CGTWDNDL 1997 TGCGGAACATGGGATAACGACCTGAGTGGTTGGCTG SGWLF TTC 1174 CGTWDNDL 1998 TGCGGAACATGGGATAACGACCTGAGTGCCTGGGTG SAWVF TTC 1175 CGTWDLTL 1999 TGCGGAACATGGGATCTCACCCTGGGTGGTGTGGTG GGVVF TTC 1176 CGTWDLSL 2000 TGCGGAACATGGGATCTCAGCCTGAGTGCTGGGGTA SAGVF TTC 1177 CGTWDLSL 2001 TGCGGAACATGGGATCTCAGCCTGAAAGAATGGGTG KEWVF TTC 1178 CGTWDLSL 2002 TGCGGAACGTGGGATCTCAGCCTGGATGCTGTTGTT DAVVF TTC 1179 CGTWDLKVF 2003 TGCGGAACCTGGGACCTGAAGGTTTTC 1180 CGTWDKTL 2004 TGCGGAACATGGGATAAGACTCTGAGTGTTTGGGTG SVWVF TTC 1181 CGTWDKSL 2005 TGCGGAACATGGGATAAGAGCCTGAGTGTTTGGGTG SVWVF TTC 1182 CGTWDKSL 2006 TGCGGAACATGGGATAAGAGCCTGAGTGGTGTGGTA SGVVF TTT 1183 CGTWDKSL 2007 TGCGGAACATGGGATAAGAGCCTGAGTGATTGGGTG SDWVF TTC 1184 CGTWDKSL 2008 TGCGGAACATGGGATAAGAGCCTGAGTGCTTTGGTT SALVF TTC 1185 CGTWDKSL 2009 TGCGGAACATGGGATAAGAGCCTGAGTGCTGGCGTC SAGVF TTC 1186 CGTWDKSL 2010 TGCGGAACATGGGATAAGAGCCTGAGTGCCGACGTC SADVF TTC 1187 CGTWDKRL 2011 TGCGGAACATGGGATAAACGCCTGACTATTGTGGTC TIVVF TTC 1188 CGTWDKRL 2012 TGCGGAACATGGGATAAACGCCTGAGTGCCTGGGTG SAWVL CTC 1189 CGTWDKNL 2013 TGCGGAACATGGGATAAGAACCTGCGTGCTGTGGTC RAVVF TTC 1190 CGTWDITLS 2014 TGCGGAACATGGGATATCACCCTGAGTGGGTTTGTC GFVF TTC 1191 CGTWDITL 2015 TGCGGAACATGGGATATCACCTTGCATACTGGAGTA HTGVF TTC 1192 CGTWDISV 2016 TGCGGAACATGGGATATCAGTGTGACTGTGGTGTTC TVVF 1193 CGTWDISV 2017 TGCGGAACATGGGATATCAGTGTGAGGGGTTATGCC RGYAF TTC 1194 CGTWDISR 2018 TGCGGAACATGGGATATCAGCCGTTGGGTTTTC WVF 1195 CGTWDISPS 2019 TGCGGAACATGGGATATCAGCCCGAGTGCTTGGGTG AWVF TTC 1196 CGTWDISLS 2020 TGCGGAACATGGGATATTAGCCTGAGTGTCTGGGTG VWVF TTC 1197 CGTWDISLS 2021 TGCGGAACATGGGATATCAGCCTGAGTGTTGTATTC VVF 1198 CGTWDISLS 2022 TGCGGAACTTGGGATATCAGCCTGAGTTCTGTGGTG SVVF TTC 1199 CGTWDISLS 2023 TGCGGAACATGGGATATCAGCCTGAGTCACTGGTTG HWLF TTC 1200 CGTWDISLS 2024 TGCGGAACATGGGATATCAGTCTGAGTGGTTGGGTG GWVF TTC 1201 CGTWDISLS 2025 TGCGGAACATGGGATATCAGCCTGAGTGGTCGAGTG GRVF TTC 1202 CGTWDISLS 2026 TGCGGAACATGGGACATCAGCCTGAGTGCTTGGGCG AWAF TTC 1203 CGTWDISLS 2027 TGCGGAACATGGGATATCAGCCTGAGTGCTGTGGTT AVVF TTC 1204 CGTWDISLS 2028 TGCGGGACATGGGACATCAGCCTGAGTGCTGTGATA AVIF TTC 1205 CGTWDISLS 2029 TGCGGAACATGGGATATCAGCCTGAGTGCTGTGTTC AVF 1206 CGTWDISLS 2030 TGCGGAACATGGGATATCAGCCTGAGTGCCCGGGTG ARVF TTC 1207 CGTWDISLS 2031 TGCGGAACATGGGATATCAGCCTGAGTGCCCTGGTG ALVF TTC 1208 CGTWDISLS 2032 TGCGGAACATGGGATATTAGCCTGAGTGCCCATGTC AHVF TTC 1209 CGTWDISLS 2033 TGCGGAACATGGGATATCAGCCTGAGTGCTGGGGTG AGVVF GTATTC 1210 CGTWDISLS 2034 TGCGGAACATGGGATATCAGCCTGAGTGCCGGCCCT AGPYVF TATGTCTTC 1211 CGTWDISLS 2035 TGCGGCACATGGGATATCAGCCTGAGTGCTGGAGGG AGGVF GTGTTC 1212 CGTWDISLS 2036 TGCGGAACATGGGATATCAGCCTGAGTGCTGAGGTT AEVF TTC 1213 CGTWDISLS 2037 TGCGGAACATGGGATATCAGCCTGAGTGCTGCTGTG AAVF TTC 1214 CGTWDISL 2038 TGCGGAACATGGGATATCAGCCTGCGTGCTGTGTTC RAVF 1215 CGTWDISL 2039 TGCGGAACATGGGATATTAGCCTGAATACTGGGGTG NTGVF TTC 1216 CGTWDISL 2040 TGCGGAACATGGGATATCAGCCTAAATAATTATGTC NNYVF TTC 1217 CGTWDISLI 2041 TGCGGAACATGGGATATCAGCCTAATTGCTGGGGTA AGVF TTC 1218 CGTWDISL 2042 TGCGGAACATGGGATATCAGCCTGCATACTTGGCTG HTWLF TTC 1219 CGTWDIRL 2043 TGCGGAACATGGGATATCCGCCTGACCGATGAGCTG TDELLF TTATTC 1220 CGTWDIRL 2044 TGCGGAACATGGGATATCAGACTGAGCGGTTTTGTT SGFVF TTC 1221 CGTWDINL 2045 TGCGGAACATGGGATATCAACCTGGGTGCTGGGGGC GAGGLYVF CTTTATGTCTTC 1222 CGTWDIILS 2046 TGCGGAACATGGGATATCATCCTGAGTGCTGAGGTA AEVF TTC 1223 CGTWDHTL 2047 TGCGGAACATGGGATCACACCCTGAGTGCTGTCTTC SAVF 1224 CGTWDHTL 2048 TGCGGAACATGGGACCACACTCTGCTTACTGTGTTA LTVLF TTC 1225 CGTWDHSL 2049 TGCGGAACATGGGATCACAGCCTGACTGCTGTGGTA TAVVF TTC 1226 CGTWDHSL 2050 TGCGGAACCTGGGATCACAGCCTGACTGCTGGGATA TAGIF TTC 1227 CGTWDHSL 2051 TGCGGAACATGGGATCACAGCCTGAGTGTTGTATTA SVVLF TTC 1228 CGTWDHSL 2052 TGCGGAACATGGGATCACAGCCTGAGTTTGGTATTC SLVF 1229 CGTWDHSL 2053 TGCGGAACATGGGATCACAGCCTGTCTATTGGGGTT SIGVF TTC 1230 CGTWDHSL 2054 TGCGGAACATGGGATCACAGCCTGAGTGCTGGGGTG SAGVF TTC 1231 CGTWDHSL 2055 TGTGGAACTTGGGATCACAGCCTGAGTGCTTTCGTG SAFVF TTC 1232 CGTWDHSL 2056 TGCGGAACATGGGATCACAGTCTGAGTGCTGCTGTT SAAVF TTC 1233 CGTWDHNL 2057 TGCGGAACATGGGACCACAATCTGCGTGCTGTCTTC RAVF 1234 CGTWDFTL 2058 TGCGGGACATGGGATTTCACCCTGAGTGTTGGGCGC SVGRF TTC 1235 CGTWDFTL 2059 TGCGGAACATGGGATTTCACCCTGAGTGCTCCTGTC SAPVF TTC 1236 CGTWDFSV 2060 TGCGGAACGTGGGATTTCAGCGTGAGTGCTGGGTGG SAGWVF GTGTTC 1237 CGTWDFSL 2061 TGCGGAACGTGGGATTTCAGTCTTACTACCTGGTTAT TTWLF TC 1238 CGTWDFSL 2062 TGCGGAACATGGGATTTCAGCCTGAGTGTTTGGGTG SVWVF TTC 1239 CGTWDFSL 2063 TGCGGAACATGGGATTTCAGCCTGAGTACTGGGGTT STGVF TTC 1240 CGTWDFSL 2064 TGCGGCACATGGGATTTCAGCCTGAGTGGTGTGGTA SGVVF TTC 1241 CGTWDFSL 2065 TGCGGAACATGGGATTTCAGCCTGAGTGGTTTCGTG SGFVF TTC 1242 CGTWDFSL 2066 TGCGGAACATGGGATTTCAGCCTGAGTGCTGGGGTG SAGVF TTC 1243 CGTWDETV 2067 TGCGGAACATGGGATGAAACCGTGAGAGGTTGGGT RGWVF GTTC 1244 CGTWDESL 2068 TGCGGAACATGGGATGAAAGTCTGAGAAGCTGGGT RSWVF GTTC 1245 CGTWDERQ 2069 TGCGGAACTTGGGATGAGAGGCAGACTGATGAGTCC TDESYVF TATGTCTTC 1246 CGTWDERL 2070 TGCGGAACATGGGATGAGAGACTCGTTGCTGGCCAG VAGQVF GTCTTC 1247 CGTWDERL 2071 TGCGGAACATGGGATGAGAGACTGAGTCCTGGAGCT SPGAFF TTTTTC 1248 CGTWDEKVF 2072 TGCGGAACATGGGATGAGAAGGTGTTC 1249 CGTWDEGQ 2073 TGCGGAACCTGGGATGAAGGCCAGACTACTGATTTC TTDFFVF TTTGTCTTC 1250 CGTWDDTL 2074 TGCGGAACATGGGATGACACCCTGGCTGGTGTGGTC AGVVF TTC 1251 CGTWDDRL 2075 TGCGGAACATGGGATGACAGGCTGACTTCTGCGGTC TSAVF TTC 1252 CGTWDDRL 2076 TGCGGAACATGGGATGACAGACTGTTTGTTGTGGTA FVVVF TTC 1253 CGTWDDNL 2077 TGCGGAACATGGGATGATAACCTGAGAGGTTGGGTG RGWVF TTC 1254 CGTWDDNL 2078 TGCGGAACATGGGATGACAACCTGCGTGGTGTCGTG RGVVF TTC 1255 CGTWDDNL 2079 TGCGGAACCTGGGATGACAATTTGAATATTGGAAGG NIGRVF GTGTTC 1256 CGTWDDIL 2080 TGCGGAACATGGGATGACATCCTGAGTGCTGTGATA SAVIF TTC 1257 CGTWDDIL 2081 TGCGGAACATGGGATGATATCCTGAGAGGTTGGGTG RGWVF TTC 1258 CGTWDATL 2082 TGCGGAACATGGGATGCCACCCTGAGTCCTGGGTGG SPGWLF TTATTC 1259 CGTWDASV 2083 TGCGGAACATGGGATGCCAGCGTGACTTCTTGGGTG TSWVF TTC 1260 CGTWDASL 2084 TGCGGAACATGGGATGCCAGCCTGACTTCTGTGGTC TSVVF TTC 1261 CGTWDASL 2085 TGCGGAACATGGGATGCCAGCCTGAGTGTTTGGGTG SVWVF TTC 1262 CGTWDASL 2086 TGCGGAACATGGGATGCCAGCCTGAGTGTTCCTTGG SVPWVF GTGTTC 1263 CGTWDASL 2087 TGCGGAACATGGGATGCCAGCCTGAGTGTGGCGGTA SVAVF TTC 1264 CGTWDASL 2088 TGCGGAACATGGGATGCCAGCCTGAGTACCTGGGTA STWVF TTC 1265 CGTWDASL 2089 TGCGGAACATGGGATGCCAGCCTGAGTGGTGTGGTA SGVVF TTC 1266 CGTWDASL 2090 TGCGGAACATGGGATGCCAGCCTGAGTGGTGGGGG SGGGEF AGAATTC 1267 CGTWDASL 2091 TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGGTG SAGVF TTC 1268 CGTWDASL 2092 TGCGGAACATGGGATGCCAGCCTGAGTGCTGGGCTT SAGLF TTC 1269 CGTWDASL 2093 TGTGGCACATGGGATGCCAGCCTGAGTGCTGAAGTC SAEVF TTC 1270 CGTWDASL 2094 TGCGGAACATGGGATGCCAGCCTGAGTGCTGACTTT SADFWVF TGGGTGTTC 1271 CGTWDASL 2095 TGCGGAACATGGGATGCCAGCCTGAGAGTCTTCTTC RVFF 1272 CGTWDASL 2096 TGCGGAACATGGGATGCCAGTCTGAGGGCTGTGGTA RAVVL CTC 1273 CGTWDASL 2097 TGCGGAACATGGGATGCCAGCCTGAATATTTGGGTT NIWVF TTC 1274 CGTWDASL 2098 TGCGGGACATGGGATGCCAGCCTGAAGAATCTGGTC KNLVF TTC 1275 CGTWDASL 2099 TGCGGAACATGGGATGCCAGCCTGGGTGCCTGGGTA GAWVF TTC 1276 CGTWDASL 2100 TGCGGAACATGGGATGCCAGCCTGGGTGCTGTGGTC GAVVF TTC 1277 CGTWDASL 2101 TGCGGAACATGGGATGCCAGCCTGGGTGCGGGGGTC GAGVF TTC 1278 CGTWDARL 2102 TGCGGAACATGGGATGCTAGGCTGAGTGGCCTTTAT SGLYVF GTCTTC 1279 CGTWDARL 2103 TGTGGAACCTGGGATGCGAGACTGGGTGGTGCAGTC GGAVF TTC 1280 CGTWDANL 2104 TGCGGAACATGGGATGCCAATCTGCGTGCTGGGGTC RAGVF TTC 1281 CGTWDAIIS 2105 TGCGGAACATGGGATGCTATCATAAGTGGTTGGGTG GWVF TTC 1282 CGTWDAG 2106 TGCGGAACATGGGATGCCGGCCAGAGTGTTTGGGTG QSVWVF TTC 1283 CGTWDAGL 2107 TGCGGCACATGGGATGCCGGGCTGACTGGCCTTTAT TGLYVF GTCTTC 1284 CGTWDAGL 2108 TGCGGAACTTGGGATGCCGGTCTGAGTGTTTATGTC SVYVF TTC 1285 CGTWDAGL 2109 TGCGGGACATGGGATGCCGGCCTGAGTACTGGGGTC STGVF TTC 1286 CGTWDAGL 2110 TGCGGAACATGGGATGCCGGCCTGAGTGGGGACGTT SGDVF TTC 1287 CGTWDAGL 2111 TGCGGAACATGGGATGCCGGCCTGAGTGCTGGTTAT SAGYVF GTCTTC 1288 CGTWDAGL 2112 TGCGGAACATGGGATGCCGGCCTGCGTGTTTGGGTG RVWVF TTC 1289 CGTWDAGL 2113 TGCGGAACATGGGATGCCGGCCTGAGGGAAATTTTC REIF 1290 CGTWASSL 2114 TGCGGAACATGGGCCAGCAGCCTGAGTTCTTGGGTG SSWVF TTC 1291 CGTWAGSL 2115 TGCGGAACATGGGCTGGCAGCCTGAGTGGTCATGTC SGHVF TTC 1292 CGTWAGSL 2116 TGCGGAACATGGGCTGGCAGCCTGAGTGCCGCTTGG SAAWVF GTGTTC 1293 CGTWAGSL 2117 TGCGGAACATGGGCTGGCAGCCTGAATGTTTATTGG NVYWVF GTGTTC 1294 CGTWAGNL 2118 TGCGGAACATGGGCTGGCAACCTGAGACCTAATTGG RPNWVF GTGTTC 1295 CGTRGSLG 2119 TGCGGAACAAGGGGTAGCCTGGGTGGTGCGGTGTTC GAVF 1296 CGTRDTTLS 2120 TGCGGAACAAGGGATACCACCCTGAGTGTCCCGGTG VPVF TTC 1297 CGTRDTSL 2121 TGCGGAACACGGGATACCAGCCTCAATATTGAAATC NIEIF TTC 1298 CGTRDTSL 2122 TGTGGAACACGGGATACCAGCCTGAATGATGTCTTC NDVF 1299 CGTRDTRL 2123 TGCGGAACACGGGATACCCGCCTGAGTATTGTGGTT SIVVF TTC 1300 CGTRDTILS 2124 TGCGGCACACGGGATACCATCCTGAGTGCTGAGGTG AEVF TTC 1301 CGTRDRSLS 2125 TGCGGAACACGGGATAGAAGCCTGAGTGGTTGGGT GWVF GTTC 1302 CGSWYYNV 2126 TGCGGATCATGGTATTACAATGTCTTCCTTTTC FLF 1303 CGSWHSSL 2127 TGCGGATCTTGGCATAGCAGCCTCAACCTTGTCGTCT NLVVF TC 1304 CGSWGSGL 2128 TGCGGATCATGGGGTAGTGGCCTGAGTGCCCCTTAT SAPYVF GTCTTC 1305 CGSWESGL 2129 TGCGGTTCGTGGGAAAGCGGCCTGGGTGCTTGGCTG GAWLF TTC 1306 CGSWDYGL 2130 TGCGGATCCTGGGATTACGGCCTCCTACTCTTC LLF 1307 CGSWDVSL 2131 TGCGGTTCATGGGATGTCAGCCTGACTGCTGTTTTC TAVF 1308 CGSWDVSL 2132 TGCGGATCCTGGGATGTCAGTCTCAATGTTGGCATTT NVGIF TC 1309 CGSWDTTL 2133 TGCGGATCATGGGATACCACCCTGCGTGCTTGGGTG RAWVF TTC 1310 CGSWDTSP 2134 TGCGGCTCGTGGGATACCAGCCCTGTCCGTGCTTGG VRAWVF GTGTTC 1311 CGSWDTSL 2135 TGCGGATCATGGGATACCAGCCTGAGTGTTTGGGTG SVWVF TTC 1312 CGSWDTSL 2136 TGCGGATCATGGGATACCAGCCTGAGTGCTGAGGTG SAEVF TTC 1313 CGSWDTSL 2137 TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGTG RAWVF TTC 1314 CGSWDTSL 2138 TGCGGCTCGTGGGATACCAGCCTGCGTGCTTGGGCG RAWAF TTC 1315 CGSWDTSL 2139 TGCGGATCATGGGATACCAGCCTGGATGCTAGGCTG DARLF TTC 1316 CGSWDTILL 2140 TGCGGATCATGGGATACCATCCTGCTTGTCTATGTCT VYVF TC 1317 CGSWDRW 2141 TGCGGATCATGGGATCGCTGGCAGGCTGCTGTCTTC QAAVF 1318 CGSWDRSL 2142 TGCGGATCATGGGATAGGAGCCTGAGTGGGTATGTC SGYVF TTC 1319 CGSWDRSL 2143 TGCGGATCATGGGATAGAAGCCTGAGTGCTTATGTC SAYVF TTC 1320 CGSWDRSL 2144 TGCGGATCATGGGATAGGAGCCTGAGTGCCGTGGTT SAVVF TTC 1321 CGSWDNTL 2145 TGCGGATCATGGGATAACACCTTGGGTGTTGTTCTCT GVVLF TC 1322 CGSWDNRL 2146 TGCGGATCGTGGGATAACAGACTAAGTACTGTCATC STVIF TTC 1323 CGSWDNRL 2147 TGCGGAAGCTGGGATAATCGATTGAACACTGTGATT NTVIF TTC 1324 CGSWDLSP 2148 TGCGGTTCATGGGATCTCAGCCCTGTACGTGTCCTTG VRVLVF TGTTC 1325 CGSWDLSL 2149 TGCGGATCATGGGATCTCAGCCTGAGTGCTGTCGTT SAVVF TTC 1326 CGSWDKNL 2150 TGCGGATCATGGGATAAAAACCTGCGTGCTGTGCTG RAVLF TTC 1327 CGSWDISLS 2151 TGCGGCTCATGGGATATCAGCCTGAGTGCTGGGGTG AGVF TTC 1328 CGSWDIRLS 2152 TGCGGATCATGGGATATCAGACTGAGTGCAGAGGTC AEVF TTC 1329 CGSWDIKL 2153 TGCGGATCATGGGACATCAAACTGAATATTGGGGTA NIGVF TTC 1330 CGSWDFSL 2154 TGCGGATCATGGGATTTCAGTCTCAATTATTTTGTCT NYFVF TC 1331 CGSWDASL 2155 TGCGGATCATGGGATGCCAGCCTGAGTACTGAGGTG STEVF TTC 1332 CGSWDAGL 2156 TGCGGATCCTGGGATGCCGGCCTGCGTGGCTGGGTT RGWVF TTC 1333 CGRWESSL 2157 TGCGGAAGATGGGAGAGCAGCCTGGGTGCTGTGGTT GAVVF TTC 1334 CGRWDFSL 2158 TGCGGAAGATGGGATTTTAGTCTGAGTGCTTATGTC SAYVF TTC 1335 CGQWDND 2159 TGCGGACAATGGGATAACGACCTGAGTGTTTGGGTG LSVWVF TTC 1336 CGPWHSSV 2160 TGCGGACCCTGGCATAGCAGCGTGACTAGTGGCCAC TSGHVL GTGCTC 1337 CGLWDASL 2161 TGCGGATTATGGGATGCCAGCCTGAGTGCTCCTACT SAPTWVF TGGGTGTTC 1338 CGIWHTSLS 2162 TGTGGAATATGGCACACTAGCCTGAGTGCTTGGGTG AWVF TTC 1339 CGIWDYSL 2163 TGCGGAATATGGGATTACAGCCTGGATACTTGGGTG DTWVF TTC 1340 CGIWDTSLS 2164 TGCGGCATATGGGATACCAGCCTGAGTGCTTGGGTG AWVF TTC 1341 CGIWDTRL 2165 TGCGGAATTTGGGATACCAGGCTGAGTGTTTATGTC SVYVF TTC 1342 CGIWDTRL 2166 TGCGGAATTTGGGATACCAGGCTGAGTGTTTATATC SVYIF TTC 1343 CGIWDTNL 2167 TGTGGAATATGGGATACGAATCTGGGTTATCTCTTC GYLF 1344 CGIWDTGL 2168 TGCGGTATATGGGATACCGGCCTGAGTGCTGTGGTA SAVVF TTC 1345 CGIWDRSLS 2169 TGCGGAATATGGGATCGCAGCCTGAGTGCTTGGGTG AWVF TTT 1346 CGIRDTRLS 2170 TGCGGAATTCGGGATACCAGGCTGAGTGTTTATGTC VYVF TTC 1347 CGGWSSRL 2171 TGCGGAGGATGGAGTAGCAGACTGGGTGTTGGCCCA GVGPVF GTGTTT 1348 CGGWGSGL 2172 TGCGGAGGATGGGGTAGCGGCCTGAGTGCTTGGGTG SAWVF TTC 1349 CGGWDTSL 2173 TGCGGAGGATGGGATACCAGCCTGAGTGCTTGGGTG SAWVF TTC 1350 CGGWDRGL 2174 TGCGGAGGATGGGATAGGGGCCTGGATGCTTGGGTT DAWVF TTC 1351 CGAWRNN 2175 TGCGGAGCATGGCGTAATAACGTGTGGGTGTTC VWVF 1352 CGAWNRRL 2176 TGCGGAGCATGGAACAGGCGCCTGAATCCTCATTCT NPHSHWVF CATTGGGTGTTC 1353 CGAWHNK 2177 TGCGGAGCCTGGCACAACAAACTGAGCGCGGTCTTC LSAVF 1354 CGAWGSSL 2178 TGCGGAGCATGGGGTAGCAGCCTGAGAGCTAGTGTC RASVF TTC 1355 CGAWGSGL 2179 TGCGGAGCATGGGGTAGCGGCCTGAGTGCTTGGGTG SAWVF TTC 1356 CGAWESSL 2180 TGCGGAGCATGGGAAAGTAGCCTGAGTGCCCCTTAT SAPYVF GTCTTC 1357 CGAWESSL 2181 TGCGGAGCATGGGAGAGCAGCCTCAATGTTGGACTG NVGLI ATC 1358 CGAWESGR 2182 TGCGGAGCATGGGAGAGCGGCCGGAGTGCTGGGGT SAGVVF GGTGTTC 1359 CGAWDYSV 2183 TGCGGAGCTTGGGATTACAGTGTGAGTGGTTGGGTG SGWVF TTC 1360 CGAWDYSL 2184 TGCGGAGCATGGGATTACAGCCTGACTGCCGGAGTA TAGVF TTC 1361 CGAWDYRL 2185 TGCGGAGCCTGGGATTACAGACTGAGTGCCGTGCTA SAVLF TTC 1362 CGAWDVRL 2186 TGCGGAGCGTGGGATGTTCGTCTGGATGTTGGGGTG DVGVF TTC 1363 CGAWDTYS 2187 TGCGGAGCATGGGATACCTACAGTTATGTCTTC YVF 1364 CGAWDTTL 2188 TGCGGAGCATGGGATACGACCCTGAGTGGTGTGGTA SGVVF TTC 1365 CGAWDTTL 2189 TGCGGAGCGTGGGATACTACCCTGAGTGCTGTGATA SAVIF TTC 1366 CGAWDTSQ 2190 TGCGGCGCATGGGATACCAGCCAGGGTGCGTCTTAT GASYVF GTCTTT 1367 CGAWDTSP 2191 TGCGGAGCATGGGATACCAGCCCTGTACGTGCTGGG VRAGVF GTGTTC 1368 CGAWDTSL 2192 TGCGGAGCATGGGATACCAGCCTGTGGCTTTTC WLF 1369 CGAWDTSL 2193 TGCGGAGCATGGGATACCAGCCTGACTGTTTATGTC TVYVF TTC 1370 CGAWDTSL 2194 TGCGGAGCATGGGACACCAGTCTGACTGCTGGGGTG TAGVF TTC 1371 CGAWDTSL 2195 TGCGGAGCTTGGGATACCAGCCTGAGTACTGTGGTT STVVF TTC 1372 CGAWDTSL 2196 TGCGGAGCATGGGATACCAGCCTGAGTTCTAGATAC SSRYIF ATATTC 1373 CGAWDTSL 2197 TGCGGAGCATGGGATACCAGCCTGAGTGGTTATGTC SGYVF TTC 1374 CGAWDTSL 2198 TGCGGAGCCTGGGATACCAGCCTGAGTGGCTGGGTG SGWVF TTC 1375 CGAWDTSL 2199 TGCGGAGCATGGGATACCAGTCTGAGTGGTGTGCTA SGVLF TTC 1376 CGAWDTSL 2200 TGCGGAGCTTGGGATACCAGCTTGAGTGGTCTTGTT SGLVF TTC 1377 CGAWDTSL 2201 TGCGGAGCTTGGGATACCAGCTTGAGTGGTTTTGTTT SGFVF TC 1378 CGAWDTSL 2202 TGCGGAGCATGGGATACCAGCCTGAGTGGTGAGGTC SGEVF TTT 1379 CGAWDTSL 2203 TGCGGAGCTTGGGATACCAGCTTGAGTGATTTTGTTT SDFVF TC 1380 CGAWDTSL 2204 TGCGGAGCATGGGATACCAGCCTGCGAACTGCGATA RTAIF TTC 1381 CGAWDTSL 2205 TGCGGAGCATGGGATACCAGCCTGCGGCTTTTC RLF 1382 CGAWDTSL 2206 TGCGGAGCATGGGATACCAGCCTGAATGTTCATGTC NVHVF TTC 1383 CGAWDTSL 2207 TGCGGAGCATGGGATACCAGCCTCAATAAATGGGTG NKWVF TTC 1384 CGAWDTRL 2208 TGCGGAGCATGGGATACCCGCCTCAGTGCGCGGCTG SARLF TTC 1385 CGAWDTRL 2209 TGCGGAGCATGGGATACCAGACTGAGGGGTTTTATT RGFIF TTC 1386 CGAWDTNL 2210 TGCGGAGCATGGGATACTAATTTGGGGAATGTTCTC GNVLL CTC 1387 CGAWDTNL 2211 TGCGGGGCATGGGATACCAACCTGGGTAAATGGGTT GKWVF TTC 1388 CGAWDTGL 2212 TGCGGAGCATGGGATACCGGCCTTGAGTGGTATGTT EWYVF TTT 1389 CGAWDRTS 2213 TGCGGAGCATGGGATAGGACTTCTGGATTGTGGCTT GLWLF TTC 1390 CGAWDRSL 2214 TGCGGAGCGTGGGATCGTAGCCTGGTTGCTGGACTC VAGLF TTC 1391 CGAWDRSL 2215 TGCGGAGCGTGGGATAGAAGCCTGACTGTTTATGTC TVYVF TTC 1392 CGAWDRSL 2216 TGCGGAGCATGGGATAGAAGCCTGAGTGGTTATGTC SGYVF TTC 1393 CGAWDRSL 2217 TGCGGAGCATGGGATAGAAGCCTGAGTGCTTATGTC SAYVF TTC 1394 CGAWDRSL 2218 TGCGGAGCATGGGATAGAAGCCTGAGTGCGGTGGT SAVVF ATTC 1395 CGAWDRSL 2219 TGCGGAGCATGGGATCGCAGCCTGAGTGCTGGGGTT SAGVF TTC 1396 CGAWDRSL 2220 TGCGGAGCGTGGGATCGCAGCCTGCGTATTGTGGTA RIVVF TTC 1397 CGAWDRSL 2221 TGCGGAGCATGGGATAGAAGTCTGAGGGCTTACGTC RAYVF TTC 1398 CGAWDRSL 2222 TGCGGAGCATGGGATAGAAGTCTGAATGTTTGGCTG NVWLF TTC 1399 CGAWDRGL 2223 TGCGGCGCCTGGGATAGGGGCCTGAATGTCGGTTGG NVGWLF CTTTTC 1400 CGAWDNRL 2224 TGCGGCGCATGGGATAATAGACTGAGTATTTTGGCC SILAF TTC 1401 CGAWDND 2225 TGCGGAGCTTGGGATAATGACCTGACAGCTTATGTC LTAYVF TTC 1402 CGAWDFSL 2226 TGCGGGGCATGGGATTTCAGCCTGACTCCTCTCTTC TPLF 1403 CGAWDDY 2227 TGCGGAGCCTGGGATGACTATCGGGGTGTGAGTATT RGVSIYVF TATGTCTTC 1404 CGAWDDRP 2228 TGTGGAGCATGGGATGACCGGCCTTCGAGTGCCGTG SSAVVF GTTTTC 1405 CGAWDDRL 2229 TGCGGAGCATGGGATGACAGACTGACTGTCGTTGTT TVVVF TTC 1406 CGAWDDRL 2230 TGCGGAGCGTGGGATGACAGGCTGGGTGCTGTGTTC GAVF 1407 CGAWDASL 2231 TGCGGAGCGTGGGATGCCAGCCTGAATCCTGGCCGG NPGRAF GCATTC 1408 CGAWDAG 2232 TGCGGAGCATGGGATGCCGGCCTGAGGGAAATTTTC LREIF 1409 CGAWAGSP 2233 TGCGGAGCTTGGGCTGGCAGTCCGAGTCCTTGGGTT SPWVF TTC 1410 CGAFDTTLS 2234 TGCGGAGCATTCGACACCACCCTGAGTGCTGGCGTT AGVF TTC 1411 CETWESSLS 2235 TGCGAAACATGGGAGAGCAGCCTGAGTGTTGGGGTC VGVF TTC 1412 CETWESSL 2236 TGCGAAACATGGGAAAGCAGCCTGAGGGTTTGGGT RVWVF GTTC 1413 CETWDTSL 2237 TGCGAAACGTGGGATACCAGCCTGAGTGGTGGGGTG SGGVF TTC 1414 CETWDTSL 2238 TGCGAAACATGGGATACCAGCCTGAGTGACTTTTAT SDFYVF GTCTTC 1415 CETWDTSL 2239 TGCGAAACATGGGATACCAGCCTGAGTGCCCTCTTC SALF 1416 CETWDTSL 2240 TGCGAAACATGGGATACCAGCCTGCGTGCTGAAGTC RAEVF TTC 1417 CETWDTSL 2241 TGCGAAACATGGGATACCAGCCTGAATGTTGTGGTA NVVVF TTC 1418 CETWDTSL 2242 TGCGAAACATGGGATACCAGCCTGGGTGCCGTGGTG GAVVF TTC 1419 CETWDRSL 2243 TGCGAAACATGGGATAGAAGCCTGAGTGGTGTGGTA SGVVF TTC 1420 CETWDRSL 2244 TGCGAAACATGGGATAGGAGCCTGAGTGCTTGGGTG SAWVF TTT 1421 CETWDRSL 2245 TGCGAAACATGGGATCGCAGCCTGAGTGCTGTGGTC SAVVF TTC 1422 CETWDRGL 2246 TGCGAGACGTGGGATAGAGGCCTGAGTGTTGTGGTT SVVVF TTC 1423 CETWDRGL 2247 TGCGAAACATGGGATAGGGGCCTGAGTGCAGTGGT SAVVF ATTC 1424 CETWDHTL 2248 TGCGAAACATGGGATCACACCCTGAGTGTTGTGATA SVVIF TTC 1425 CETWDASL 2249 TGCGAAACATGGGATGCCAGCCTGACTGTTGTGTTA TVVLF TTC 1426 CETWDASL 2250 TGCGAAACATGGGATGCCAGCCTGAGTGCTGGGGTG SAGVF TTC 1427 CETWDAGL 2251 TGCGAAACGTGGGATGCCGGCCTGAGTGAGGTGGTG SEVVF TTC 1428 CETFDTSLS 2252 TGCGAAACATTTGATACCAGCCTGAGTGTTGTAGTC VVVF TTC 1429 CETFDTSLN 2253 TGCGAAACATTTGATACCAGCCTAAATATTGTAGTC IVVF TTT 1430 CESWDRSRI 2254 TGCGAATCATGGGATAGAAGCCGGATTGGTGTGGTC GVVF TTC 1431 CESWDRSL 2255 TGCGAAAGTTGGGACAGGAGTCTGAGTGCCCGGGTG SARVY TAC 1432 CESWDRSL 2256 TGCGAATCCTGGGATAGGAGCCTGCGTGCCGTGGTC RAVVF TTC 1433 CESWDRSLI 2257 TGCGAATCTTGGGATCGTAGTTTGATTGTGGTGTTC VVF 1434 CESWDNNL 2258 TGCGAAAGTTGGGATAACAATTTAAATGAGGTGGTT NEVVF TTC 1435 CEIWESSPS 2259 TGCGAAATATGGGAGAGCAGCCCGAGTGCTGACGA ADDLVF TTTGGTGTTC 1436 CEAWDTSL 2260 TGCGAAGCATGGGATACCAGCCTGAGTGGTGCGGTG SGAVF TTC 1437 CEAWDTSL 2261 TGCGAAGCATGGGATACCAGCCTGAGTGCCGGGGTG SAGVF TTC 1438 CEAWDTSL 2262 TGCGAAGCATGGGATACCAGCCTGGGTGGTGGGGTG GGGVF TTC 1439 CEAWDRSL 2263 TGCGAAGCATGGGATCGCAGCCTGACTGGTAGCCTG TGSLF TTC 1440 CEAWDRGL 2264 TGCGAAGCGTGGGATAGGGGCCTGAGTGCAGTGGT SAVVF ATTC 1441 CEAWDNIL 2265 TGCGAAGCCTGGGATAACATCCTGAGTACTGTGGTG STVVF TTC 1442 CEAWDISLS 2266 TGCGAAGCATGGGACATCAGCCTGAGTGCTGGGGTG AGVF TTC 1443 CEAWDADL 2267 TGCGAAGCATGGGATGCCGACCTGAGTGGTGCGGTG SGAVF TTC 1444 CATWTGSF 2268 TGCGCAACATGGACTGGTAGTTTCAGAACTGGCCAT RTGHYVF TATGTCTTC 1445 CATWSSSP 2269 TGCGCAACATGGAGTAGCAGTCCCAGGGGGTGGGT RGWVF GTTC 1446 CATWHYSL 2270 TGCGCAACATGGCATTACAGCCTGAGTGCTGGCCGA SAGRVF GTGTTC 1447 CATWHTSL 2271 TGCGCAACATGGCATACCAGCCTGAGTATTGTGCAG SIVQF TTC 1448 CATWHSTL 2272 TGCGCAACATGGCATAGCACCCTGAGTGCTGATGTG SADVLF CTTTTC 1449 CATWHSSL 2273 TGCGCAACATGGCATAGCAGCCTGAGTGCTGGCCGA SAGRLF CTCTTC 1450 CATWHIAR 2274 TGCGCAACATGGCATATCGCTCGGAGTGCCTGGGTG SAWVF TTC 1451 CATWGSSQ 2275 TGCGCAACATGGGGTAGTAGTCAGAGTGCCGTGGTA SAVVF TTC 1452 CATWGSSL 2276 TGCGCAACATGGGGTAGCAGCCTGAGTGCTGGGGGT SAGGVF GTTTTC 1453 CATWEYSL 2277 TGTGCAACATGGGAATACAGCCTGAGTGTTGTGCTG SVVLF TTC 1454 CATWETTR 2278 TGCGCAACATGGGAGACCACCCGACGTGCCTCTTTT RASFVF GTCTTC 1455 CATWETSL 2279 TGCGCAACATGGGAGACCAGCCTGAATGTTTATGTC NVYVF TTC 1456 CATWETSL 2280 TGCGCAACATGGGAAACTAGCCTGAATGTTGTGGTC NVVVF TTC 1457 CATWETSL 2281 TGCGCAACATGGGAGACCAGCCTGAATCTTTATGTC NLYVF TTC 1458 CATWETGL 2282 TGCGCAACATGGGAGACTGGCCTAAGTGCTGGAGA SAGEVF GGTGTTC 1459 CATWESTL 2283 TGCGCGACGTGGGAGAGTACCCTAAGTGTTGTGGTT SVVVF TTC 1460 CATWESSL 2284 TGCGCAACGTGGGAGAGCAGCCTGAGTATTTTTGTC SIFVF TTC 1461 CATWESSL 2285 TGCGCAACATGGGAAAGCAGCCTCAACACTTTTTAT NTFYVF GTCTTC 1462 CATWESRV 2286 TGCGCAACATGGGAGAGTAGGGTGGATACTCGAGG DTRGLLF GTTGTTATTC 1463 CATWESGL 2287 TGCGCAACATGGGAGAGCGGCCTGAGTGGTGCGGG SGAGVF GGTGTTC 1464 CATWEGSL 2288 TGCGCAACATGGGAAGGCAGCCTCAACACTTTTTAT NTFYVF GTCTTC 1465 CATWDYSL 2289 TGCGCAACTTGGGATTATAGCCTGAGTGCTGTGGTG SAVVF TTC 1466 CATWDYRL 2290 TGCGCAACATGGGATTACAGACTGAGTATTGTGGTA SIVVF TTC 1467 CATWDYNL 2291 TGCGCAACATGGGATTATAACCTGGGAGCTGCGGTG GAAVF TTC 1468 CATWDVTL 2292 TGCGCCACATGGGATGTCACCCTGGGTGTCTTGCAT GVLHF TTC 1469 CATWDTTL 2293 TGCGCAACATGGGATACAACACTGAGTGTCTGGGTC SVWVF TTC 1470 CATWDTTL 2294 TGCGCAACATGGGATACCACCCTGAGTGTAGTACTT SVVLF TTC 1471 CATWDTTL 2295 TGCGCAACATGGGATACCACCCTGAGTGTTGAGGTC SVEVF TTC 1472 CATWDTSP 2296 TGCGCAACATGGGATACCAGCCCCAGCCTGAGTGGT SLSGFWVF TTTTGGGTGTTC 1473 CATWDTSL 2297 TGCGCAACATGGGATACCAGCCTGACTGGTGTGGTA TGVVF TTC 1474 CATWDTSL 2298 TGCGCAACATGGGATACCAGCCTGACTGGTGCGGTG TGAVF TTC 1475 CATWDTSL 2299 TGCGCAACATGGGATACCAGCCTGACTGCCTGGGTA TAWVF TTC 1476 CATWDTSL 2300 TGCGCAACATGGGATACCAGCCTGACTGCTGTGGTT TAVVF TTC 1477 CATWDTSL 2301 TGCGCAACATGGGATACTAGCCTGACTGCTAAGGTG TAKVF TTC 1478 CATWDTSL 2302 TGCGCAACATGGGACACCAGCCTGAGTGTTGTGGTT SVVVF TTC 1479 CATWDTSL 2303 TGCGCTACTTGGGATACCAGCCTGAGTGTTGGGGTA SVGVF TTT 1480 CATWDTSL 2304 TGCGCAACATGGGATACCAGCCTGAGTTCTTGGGTG SSWVF TTC 1481 CATWDTSL 2305 TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTA SGGVL CTC 1482 CATWDTSL 2306 TGCGCAACATGGGATACCAGCCTGAGTGGTGGGGTG SGGVF TTC 1483 CATWDTSL 2307 TGCGCAACATGGGATACCAGCCTGAGTGGTGGCCGA SGGRVF GTGTTC 1484 CATWDTSL 2308 TGCGCAACATGGGATACCAGCCTGAGTGGTGACCGA SGDRVF GTGTTC 1485 CATWDTSL 2309 TGCGCAACGTGGGATACTAGCCTGAGTGAAGGGGTG SEGVF TTC 1486 CATWDTSL 2310 TGCGCAACCTGGGATACCAGCCTGAGTGCCGTGGTG SAVVL CTC 1487 CATWDTSL 2311 TGCGCAACATGGGATACCAGCCTGAGTGCTGTCTTC SAVF 1488 CATWDTSL 2312 TGCGCGACATGGGATACCAGCCTGAGTGCTCGGGTG SARVF TTC 1489 CATWDTSL 2313 TGCGCAACATGGGATACCAGCCTGAGTGCCTTATTC SALF 1490 CATWDTSL 2314 TGCGCAACATGGGATACCAGCCTGAGTGCTCATGTC SAHVF TTC 1491 CATWDTSL 2315 TGCGCAACATGGGATACCAGCCTGAGTGCTGGCCGG SAGRVF GTGTTC 1492 CATWDTSL 2316 TGCGCAACATGGGATACCAGCCTGAGTGCGGAGGTC SAEVF TTC 1493 CATWDTSL 2317 TGCGCAACATGGGATACCAGCCTGAGTGCTGATGCT SADAGGGVF GGTGGGGGGGTCTTC 1494 CATWDTSL 2318 TGCGCAACATGGGATACCAGCCTGCGTGTCGTGGTA RVVVF TTC 1495 CATWDTSL 2319 TGCGCAACATGGGATACCAGCCTGAGAGGGGTGTTC RGVF 1496 CATWDTSL 2320 TGCGCAACATGGGATACCAGCCTGCCTGCGTGGGTG PAWVF TTC 1497 CATWDTSL 2321 TGTGCAACATGGGATACCAGCCTGAATGTTGGGGTA NVGVF TTC 1498 CATWDTSL 2322 TGCGCAACATGGGATACCAGCCTGGGTATTGTGTTA GIVLF TTT 1499 CATWDTSL 2323 TGCGCAACATGGGACACCAGCCTGGGTGCGCGTGTG GARVVF GTCTTC 1500 CATWDTSL 2324 TGTGCAACGTGGGATACCAGTCTAGGTGCCTTGTTC GALF 1501 CATWDTSL 2325 TGCGCAACATGGGATACCAGCCTGGCGACTGGACTG ATGLF TTC 1502 CATWDTSL 2326 TGCGCAACATGGGATACCAGCCTGGCTGCCTGGGTA AAWVF TTC 1503 CATWDTRL 2327 TGCGCAACCTGGGATACCAGGCTGAGTGCTGTGGTC SAVVF TTC 1504 CATWDTRL 2328 TGCGCAACATGGGATACCAGGCTGAGTGCTGGGGTG SAGVF TTC 1505 CATWDTRL 2329 TGTGCAACGTGGGACACACGTCTACTTATTACGGTT LITVF TTC 1506 CATWDTLL 2330 TGCGCAACATGGGACACCCTCCTGAGTGTTGAACTC SVELF TTC 1507 CATWDTGR 2331 TGCGCAACATGGGATACTGGCCGCAATCCTCATGTG NPHVVF GTCTTC 1508 CATWDTGL 2332 TGCGCAACATGGGATACCGGCCTGTCTTCGGTGTTG SSVLF TTC 1509 CATWDTGL 2333 TGCGCAACGTGGGATACCGGCCTGAGTGCGGTTTTC SAVF 1510 CATWDRTL 2334 TGCGCTACGTGGGATAGGACCCTGAGTATTGGAGTC SIGVF TTC 1511 CATWDRSV 2335 TGCGCAACGTGGGATCGCAGTGTGACTGCTGTGCTC TAVLF TTC 1512 CATWDRSL 2336 TGCGCAACCTGGGATAGGAGCCTGAGTGGTGTGGTG SGVVF TTC 1513 CATWDRSL 2337 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTGGTC SAVVF TTC 1514 CATWDRSL 2338 TGCGCAACATGGGATAGAAGCCTGAGTGCTGTTCCT SAVPWVF TGGGTGTTC 1515 CATWDRSL 2339 TGCGCAACATGGGATCGCAGCCTGAGTGCTGGGGTG SAGVF TTC 1516 CATWDRSL 2340 TGCGCAACGTGGGATAGGAGCCTGCGTGCTGGGGTG RAGVF TTC 1517 CATWDRSL 2341 TGCGCAACATGGGATCGCAGTCTGAATGTTTATGTC NVYVL CTC 1518 CATWDRIL 2342 TGCGCAACGTGGGATCGCATCCTGAGCGCTGAGGTG SAEVF TTC 1519 CATWDRGL 2343 TGCGCAACGTGGGATAGAGGCCTGAGTACTGGGGTG STGVF TTC 1520 CATWDNYL 2344 TGCGCAACATGGGATAACTACCTGGGTGCTGCCGTG GAAVF TTC 1521 CATWDNTP 2345 TGCGCAACATGGGATAACACGCCTTCGAATATTGTG SNIVVF GTATTC 1522 CATWDNTL 2346 TGCGCAACATGGGATAATACACTGAGTGTGTGGGTC SVWVF TTC 1523 CATWDNTL 2347 TGCGCAACATGGGATAACACCCTGAGTGTCAATTGG SVNWVF GTGTTC 1524 CATWDNTL 2348 TGCGCAACCTGGGATAACACACTGAATGTCTTTTAT NVFYVF GTTTTC 1525 CATWDNRL 2349 TGTGCGACATGGGATAATCGGCTCAGTTCTGTGGTC SSVVF TTC 1526 CATWDNRL 2350 TGCGCAACATGGGATAACCGCCTGAGTGCTGGGGTG SAGVL CTC 1527 CATWDNRL 2351 TGCGCAACGTGGGATAACAGGCTGAGTGCTGGGGTG SAGVF TTC 1528 CATWDNRD 2352 TGCGCAACATGGGATAACAGGGATTGGGTCTTC WVF 1529 CATWDNNL 2353 TGCGCAACATGGGATAACAACCTGGGTGCTGGGGTG GAGVF TTC 1530 CATWDNKL 2354 TGCGCAACATGGGATAACAAGCTGACTTCTGGGGTC TSGVF TTC 1531 CATWDNIL 2355 TGCGCAACATGGGATAACATCCTGAGTGCCTGGGTG SAWVF TTT 1532 CATWDNDI 2356 TGCGCAACCTGGGACAACGATATACATTCTGGGCTG HSGLF TTC 1533 CATWDLSL 2357 TGCGCAACTTGGGATCTCAGCCTGAGTGCCCTGTTC SALF 1534 CATWDITLS 2358 TGCGCAACATGGGATATCACCCTGAGTGCTGAGGTG AEVF TTC 1535 CATWDISPS 2359 TGCGCAACGTGGGATATCAGCCCGAGTGCTGGCGGG AGGVF GTGTTC 1536 CATWDISLS 2360 TGCGCAACATGGGATATCAGTCTAAGTACTGGCCGG TGRAVF GCTGTGTTC 1537 CATWDISLS 2361 TGCGCAACATGGGATATCAGTCTGAGTCAGGTATTC QVF 1538 CATWDIRL 2362 TGCGCAACATGGGATATCAGGCTGAGTAGTGGAGTG SSGVF TTC 1539 CATWDIGP 2363 TGCGCAACGTGGGATATCGGCCCGAGTGCTGGCGGG SAGGVF GTGTTC 1540 CATWDHSR 2364 TGCGCAACATGGGATCACAGCCGGGCTGGTGTGCTA AGVLF TTC 1541 CATWDHSP 2365 TGCGCAACATGGGATCACAGTCCGAGTGTTGGAGAA SVGEVF GTCTTC 1542 CATWDHSL 2366 TGCGCAACATGGGATCACAGCCTGCGTGTTGGGGTG RVGVF TTC 1543 CATWDHSL 2367 TGCGCAACATGGGATCACAGCCTGAACATTGGGGTG NIGVF TTC 1544 CATWDHSL 2368 TGCGCAACATGGGATCACAGCCTGGGTCTTTGGGCA GLWAF TTC 1545 CATWDHNL 2369 TGCGCCACATGGGATCACAATCTGCGTCTTGTTTTC RLVF 1546 CATWDHIL 2370 TGCGCGACTTGGGATCACATCCTGGCTTCTGGGGTG ASGVF TTC 1547 CATWDFSL 2371 TGCGCAACATGGGATTTCAGCCTGAGTGTTTGGGTG SVWVF TTC 1548 CATWDFSL 2372 TGCGCAACATGGGATTTCAGCCTGAGTGCTTGGGTG SAWVF TTC 1549 CATWDDTL 2373 TGCGCAACATGGGATGACACCCTCACTGCTGGTGTG TAGVF TTC 1550 CATWDDRL 2374 TGCGCAACATGGGACGACAGGCTGAGTGCTGTGCTT SAVLF TTC 1551 CATWDDRL 2375 TGCGCAACATGGGATGACAGGCTGGATGCTGCGGTG DAAVF TTC 1552 CATWDATL 2376 TGCGCAACATGGGATGCGACCCTGAATACTGGGGTG NTGVF TTC 1553 CATWDASL 2377 TGCGCAACATGGGATGCCAGCCTGAGTGTTTGGCTG SVWLL CTC 1554 CATWDASL 2378 TGCGCGACATGGGATGCCAGCCTGAGTGGTGGGGTG SGGVF TTC 1555 CATRDTTLS 2379 TGCGCAACACGGGATACCACCCTCAGCGCCGTTCTG AVLF TTC 1556 CATLGSSLS 2380 TGCGCTACATTGGGTAGTAGCCTGAGTCTCTGGGTG LWVF TTC 1557 CATIETSLP 2381 TGCGCAACAATCGAAACTAGCCTGCCTGCCTGGGTA AWVF TTC 1558 CATGDRSL 2382 TGCGCAACAGGGGACAGAAGCCTGACTGTTGAGGT TVEVF ATTC 1559 CATGDLGL 2383 TGCGCTACAGGGGATCTCGGCCTGACCATAGTCTTC TIVF 1560 CASWDYRG 2384 TGCGCATCATGGGATTACAGGGGGAGATCTGGTTGG RSGWVF GTGTTC 1561 CASWDTTL 2385 TGCGCATCATGGGATACCACCCTGAATGTTGGGGTG NVGVF TTC 1562 CASWDTTL 2386 TGCGCTTCATGGGATACCACCCTGGGTTTTGTGTTAT GFVLF TC 1563 CASWDTSL 2387 TGCGCATCATGGGATACCAGCCTGAGTGGTGGTTAT SGGYVF GTCTTC 1564 CASWDTSL 2388 TGCGCATCATGGGATACCAGCCTCCGTGCTGGGGTG RAGVF TTC 1565 CASWDTSL 2389 TGCGCATCATGGGATACCAGCCTGGGTGCTGGGGTG GAGVF TTC 1566 CASWDRGL 2390 TGCGCATCATGGGACAGAGGCCTGAGTGCAGTGGTG SAVVF TTC 1567 CASWDNVL 2391 TGTGCTAGTTGGGATAACGTCCTGCGTGGTGTGGTA RGVVF TTC 1568 CASWDNRL 2392 TGCGCGTCATGGGATAACAGGCTGACTGCCGTGGTT TAVVF TTC 1569 CASWDASL 2393 TGCGCATCATGGGATGCAAGCCTGTCCGTCGCTTTC SVAF 1570 CASWDAGL 2394 TGCGCTTCGTGGGATGCCGGCCTGAGTTCTTATGTCT SSYVF TC 1571 CASGDTSLS 2395 TGCGCATCCGGGGATACCAGCCTGAGTGGTGTGATA GVIF TTC 1572 CARWHTSL 2396 TGCGCAAGATGGCATACGAGCCTAAGTATTTGGGTC SIWVF TTC 1573 CAIWDTGL 2397 TGCGCAATATGGGATACCGGCCTGAGTCCTGGCCAA SPGQVAF GTTGCCTTC 1574 CAAWHSGL 2398 TGCGCAGCATGGCATAGCGGCCTGGGTCTCCCGGTC GLPVF TTC 1575 CAAWDYSL 2399 TGCGCAGCATGGGATTACAGCCTGAGTGCTGGGGTG SAGVF TTC 1576 CAAWDTTL 2400 TGCGCAGCCTGGGATACTACCCTGCGTGTTAGGCTG RVRLF TTC 1577 CAAWDTSL 2401 TGCGCAGCATGGGATACCAGCCTGACTGCCTGGGTT TAWVF TTC 1578 CAAWDTSL 2402 TGCGCAGCATGGGATACCAGCTTGAGTGGTGGGGTG SGGVF TTC 1579 CAAWDTSL 2403 TGCGCAGCATGGGATACCAGCCTGAGTGGCGAGGCT SGEAVF GTGTTC 1580 CAAWDTSL 2404 TGCGCAGCATGGGATACCAGCTTGAGTGGTGCGGTG SGAVF TTC 1581 CAAWDTSL 2405 TGCGCAGCATGGGATACCAGCCTGAGTGCCTGGGTG SAWVF TTC 1582 CAAWDTSL 2406 TGCGCAGCATGGGATACCAGCCTGAGTGCTGGGGTA SAGVF TTC 1583 CAAWDTSL 2407 TGCGCAGCATGGGATACCAGCCTGGATACTTATGTC DTYVF TTC 1584 CAAWDTRL 2408 TGCGCTGCATGGGATACCCGTCTGAGTGGTGTGTTA SGVLF TTC 1585 CAAWDTRL 2409 TGCGCAGCATGGGATACCAGGCTGAGTGCTGGGGTG SAGVF TTC 1586 CAAWDRSL 2410 TGCGCAGCATGGGATCGCAGTCTGAGTACTGGAGTT STGVF TTC 1587 CAAWDIRR 2411 TGCGCAGCGTGGGATATCCGCCGGTCTGTCCTTTTC SVLF 1588 CAAWDHT 2412 TGCGCTGCGTGGGATCACACTCAGCGTCTTTCCTTC QRLSF 1589 CAAWDHSL 2413 TGCGCAGCATGGGATCACAGCCTGAGTGCTGGCCAG SAGQVF GTGTTC 1590 CAAVDTGL 2414 TGCGCAGCAGTCGATACTGGTCTGAAAGAATGGGTG KEWVF TTC

The CDRs were prescreened to contain no amino acid liabilities, cryptic splice sites or nucleotide restriction sites. The CDR variation was observed in at least two individuals and comprises the near-germline space of single, double and triple mutations. The order of assembly is seen in FIG. 21C.

The VH domains that were designed include IGHV1-69 and IGHV3-30. Each of two heavy chain VH domains are assembled with their respective invariant 4 framework elements (FW1, FW2, FW3, FW4) and variable 3 CDR (H1, H2, H3) elements. For IGHV1-69, 417 variants were designed for H1 and 258 variants were designed for H2. For IGHV3-30, 535 variants were designed for H1 and 165 variants were designed for H2. For the CDR H3, the same cassette was used in both IGHV1-69 and IGHV-30 since both designed use an identical FW4, and because the edge of FW3 is also identical for both IGHV1-69 and IGHV3-30. The CDR H3 comprises an N-terminus and C-terminus element that are combinatorially joined to a central middle element to generate 1×10¹⁰ diversity. The N-terminal and middle element overlap with a “GGG” glycine codon. The middle and C-terminal element overlap with a “GGT” glycine codon. The CDR H3 comprises 5 subpools that were assembled separately. The various N-terminus and C-terminus elements comprise sequences as seen in Table 14.

TABLE 14 Sequences for N-terminus and C-terminus elements SEQ Element ID NO Sequence Stem A 2415 CARDLRELECEEWT XXX SRGPCVDPRGVAGSFD VW Stem B 2416 CARDMYYDF XXX EVVPADDAFDIW Stem C 2417 CARDGRGSLPRPKGGP XXX YDSSEDSGGAFDIW Stem D 2418 CARANQHF XXX GYHYYGMDVW Stem E 2419 CAKHMSMQ XXX RADLVGDAFDVW

Example 12. Enrichment for GPCR GLP1R Binding Proteins

Antibodies having CDR-H3 regions with a variant fragments of GPCR binding protein were generated by methods described herein were panned using cell-based methods to identified variants which are enriched for binding to particular GPCRs, as described in Example 10.

Variants of the GLP C-terminus peptide were identified (listed in Table 15) that when embedded in the CDR-H3 region of an antibody, were repeatedly and selectively enriched for binding to GPCR GLP1R.

TABLE 15 Sequences of GLP1 embedded in CDR-H3 SEQ ID NO Sequence 2420 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2421 CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2422 CAKHMSMQDYLVIGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2423 CAKHMSMQEGAVTGEGQDAKEFIAWLVKGRVRADLVGDAFDVW 2424 WAKHMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2425 CARDGRGSLPRPKGGPQTVGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2426 CARANQHFYEQEGTFTSDVSSYLEGQAAKEFIAWLVKGGIRGYHYYGMDVW 2427 CARANQHFTELHGEGQAAKEFIAWLVKGRGQIDIGYHYYGMDVW 2428 CARANQHFLGAGVSSYLEGQAAKEFIAWLVKGDTTGYHYYGMDVW 2429 CARANQHFLDKGTFTSDVSSYLEGQAAKEFIAWLVKGIYPGYHYYGMDVW 2430 CARANQHFGTLSAGEGQAAKEFIAWLVKGGSQYDSSEDSGGAFDIW 2431 CARANQHFGLHAQGEGQAAKEFIAWLVKGSGTYGYHYYGMDVW 2432 CARANQHFGGKGEGQAAKEFIAWLVKGGGSGAGYHYYGMDVW 2433 CAKQMSMQEGAVTGEGQAAKEFIAWLVKGRVRADLVGDAFDVW 2434 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGPARADLVGDAFDVW 2435 CAKHMSMQEGAVTGEGQAAKEFIAWLVKGGLTYDSSEDSGGAFDIW 2436 CAKHMSMQDYLVIGEGQAAKEFIAWLVKGRVRADLVGDAFDVW

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. 

What is claimed is:
 1. A nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid comprises: a) a nucleic acid sequence comprising a length of about 80 to about 230 base pairs that encodes for a CDR-H3 loop, wherein the CDR-H3 loop comprises a sequence of any one of SEQ ID NOs: 2420 to 2436; and b) a nucleic acid sequence encoding a variable domain of light chain (VL), wherein the nucleic acid sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs: 94, 96, 98, 283, 301, 321, 416, 432, 459, 460, 616, 618, 647, 652, 660, 666, 677, 745, 755, 1627, 1883, and 2043, wherein each of the nucleic acids encodes for a sequence that when translated encodes for an immunoglobulin scaffold that comprises a GLP1R binding domain, and wherein the GLP1R binding domain is a ligand for GLP1R.
 2. The nucleic acid library of claim 1, wherein the immunoglobulin scaffold further comprises one or more domains selected from variable domain of heavy chain (VH), constant domain of light chain (CL), and constant domain of heavy chain (CH).
 3. The nucleic acid library of claim 2, wherein a length of the VH domain is about 280 to about 300 base pairs.
 4. The nucleic acid library of claim 1, wherein a length of the VL domain is about 90 to about 120 amino acids.
 5. The nucleic acid library of claim 1, wherein a length of the VL domain is about 300 to about 350 base pairs.
 6. The nucleic acid library of claim 1, wherein the library comprises at least 10⁵ non-identical nucleic acids.
 7. The nucleic acid library of claim 1, wherein the immunoglobulin scaffold comprises a single immunoglobulin domain.
 8. The nucleic acid library of claim 1, wherein the immunoglobulin scaffold comprises a peptide of at most 100 amino acids.
 9. The nucleic acid library of claim 1, wherein the nucleic acid sequence encoding a variable domain of light chain (VL) comprises a sequence having at least about 90% sequence identity to SEQ ID NOs: 94, 96, and
 98. 10. The nucleic acid library of claim 1, wherein the nucleic acid sequence encoding a variable domain of light chain (VL) comprises a sequence having at least about 90% sequence identity to any one of SEQ ID NOs: 616, 618, 647, 652, 660, 666, 677, 745, and
 755. 